It is well described that excessive lipid metabolism can cause insulin resistance in both animals and humans, and this has been implicated as a causative factor in the development of insulin resistance and type 2 diabetes in humans. Recently, we have shown that intravenous lipid emulsion (liposyn) infusion during a 120-min euglycemic-hyperinsulinemic clamp led to significant reductions in insulin action and fatty acid translocase (FAT/CD36) skeletal muscle protein expression. After reviewing the literature, it became evident that essentially all past studies, including our own, were conducted in male animals. Therefore, to determine whether there were sex determinants of fat-induced insulin resistance, we assessed the impact of free fatty acid (FFA) elevation on insulin action in female rats. Here, we report that a fourfold elevation in plasma FFA concentration induced a 40% reduction in the insulin-stimulated glucose disposal rate, a 30% decline in insulin-stimulated skeletal muscle insulin substrate receptor-1 (IRS-1) phosphorylation, a 48% decrease in IRS-1–associated phosphatidylinositol (PI) 3-kinase activity, and a 50% reduction in muscle FAT/CD36 protein expression in male rats. In striking contrast, we found no effect of FFA elevation to cause insulin resistance, changes in IRS-1/PI 3-kinase, or FAT/CD36 protein levels in female animals. Our findings indicate that female animals are protected from lipid-induced reductions in insulin action.
Obesity and type 2 diabetes are characterized by decreased insulin sensitivity, elevated concentrations of free fatty acids (FFAs), and increased macrophage infiltration in adipose tissue (AT). Here, we show that FFAs can cause activation of RAW264.7 cells primarily via the JNK signaling cascade and that TLR2 and TLR4 are upstream of JNK and help transduce FFA proinflammatory signals. We also demonstrate that F4/80+CD11b+CD11c+ bone marrow-derived dendritic cells (BMDCs) have heightened proinflammatory activity compared with F4/80+CD11b+CD11c− bone marrow-derived macrophages and that the proinflammatory activity and JNK phosphorylation of BMDCs, but not bone marrow-derived macrophages, was further increased by FFA treatment. F4/80+CD11b+CD11c+ cells were found in AT, and the proportion and number of these cells in AT is increased in ob/ob mice and by feeding wild type mice a high fat diet for 1 and 12 weeks. AT F4/80+CD11b+CD11c+ cells express increased inflammatory markers compared with F4/80+CD11b+CD11c− cells, and FFA treatment increased inflammatory responses in these cells. In addition, we found that CD11c expression is increased in skeletal muscle of high fat diet-fed mice and that conditioned medium from FFA-treated wild type BMDCs, but not TLR2/4 DKO BMDCs, can induce insulin resistance in L6 myotubes. Together our results show that FFAs can activate CD11c+ myeloid proinflammatory cells via TLR2/4 and JNK signaling pathways, thereby promoting inflammation and subsequent cellular insulin resistance. Obesity and type 2 diabetes are characterized by decreased insulin sensitivity, elevated concentrations of free fatty acids (FFAs), and increased macrophage infiltration in adipose tissue (AT). Here, we show that FFAs can cause activation of RAW264.7 cells primarily via the JNK signaling cascade and that TLR2 and TLR4 are upstream of JNK and help transduce FFA proinflammatory signals. We also demonstrate that F4/80+CD11b+CD11c+ bone marrow-derived dendritic cells (BMDCs) have heightened proinflammatory activity compared with F4/80+CD11b+CD11c− bone marrow-derived macrophages and that the proinflammatory activity and JNK phosphorylation of BMDCs, but not bone marrow-derived macrophages, was further increased by FFA treatment. F4/80+CD11b+CD11c+ cells were found in AT, and the proportion and number of these cells in AT is increased in ob/ob mice and by feeding wild type mice a high fat diet for 1 and 12 weeks. AT F4/80+CD11b+CD11c+ cells express increased inflammatory markers compared with F4/80+CD11b+CD11c− cells, and FFA treatment increased inflammatory responses in these cells. In addition, we found that CD11c expression is increased in skeletal muscle of high fat diet-fed mice and that conditioned medium from FFA-treated wild type BMDCs, but not TLR2/4 DKO BMDCs, can induce insulin resistance in L6 myotubes. Together our results show that FFAs can activate CD11c+ myeloid proinflammatory cells via TLR2/4 and JNK signaling pathways, thereby promoting inflammation and subsequent cellular insulin resistance. Chronic inflammation is a well described feature of insulin resistance and obesity characterized by elevated proinflammatory JNK 2The abbreviations used are: JNK, c-Jun N-terminal kinase; FA, fatty acid; FFA, free fatty acid; TLR, Toll-like receptor; AT, adipose tissue; BM, bone marrow; BMDC, bone marrow-derived dendritic cell; BMDM, bone marrow-derived macrophage; HF, high fat; HFD, high fat diet; IL, interleukin; TNF, tumor necrosis factor; FBS, fetal bovine serum; BSA, bovine serum albumin; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay; siRNA, small interfering RNA; WT, wild type; GM-CSF, granulocyte macrophage-colony-stimulating factor; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; ITT, insulin tolerance test; SVF, stromal vascular fraction; SVC, stromal vascular cell; NC, normal chow; MNC, mononuclear cells; CM, conditioned medium; KD, knockdown; qPCR, quantitative PCR. and IKKβ kinase activity (1Yuan M. Konstantopoulos N. Lee J. Hansen L. Li Z.W. Karin M. Shoelson S.E. Science. 2001; 293: 1673-1677Crossref PubMed Scopus (1652) Google Scholar, 2Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2679) Google Scholar) and increased cyto/chemokine expression in insulin target tissues (3Dandona P. Aljada A. Bandyopadhyay A. Trends Immunol. 2004; 25: 4-7Abstract Full Text Full Text PDF PubMed Scopus (1672) Google Scholar). In white adipose tissue, chronic inflammation is associated with an increase in macrophage infiltration (4Weisberg S.P. McCann D. Desai M. Rosenbaum M. Leibel R.L. Ferrante Jr., A.W. J. Clin. Investig. 2003; 112: 1796-1808Crossref PubMed Scopus (7637) Google Scholar, 5Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. J. Clin. Investig. 2003; 112: 1821-1830Crossref PubMed Scopus (5275) Google Scholar, 6Bouloumie A. Curat C.A. Sengenes C. Lolmede K. Miranville A. Busse R. Curr. Opin. Clin. Nutr. Metab. Care. 2005; 8: 347-354Crossref PubMed Scopus (228) Google Scholar). Surgically induced weight loss (7Cancello R. Henegar C. Viguerie N. Taleb S. Poitou C. Rouault C. Coupaye M. Pelloux V. Hugol D. Bouillot J.L. Bouloumie A. Barbatelli G. Cinti S. Svensson P.A. Barsh G.S. Zucker J.D. Basdevant A. Langin D. Clement K. Diabetes. 2005; 54: 2277-2286Crossref PubMed Scopus (894) Google Scholar), diet and exercise (8Bruun J.M. Helge J.W. Richelsen B. Stallknecht B. Am. J. Physiol. 2006; 290: E961-E967Crossref PubMed Scopus (343) Google Scholar), and treatment with rosiglitazone, an insulin-sensitizing drug (5Xu H. Barnes G.T. Yang Q. Tan G. Yang D. Chou C.J. Sole J. Nichols A. Ross J.S. Tartaglia L.A. Chen H. J. Clin. Investig. 2003; 112: 1821-1830Crossref PubMed Scopus (5275) Google Scholar), all reduce macrophage infiltration in white adipose tissue (AT) and decrease the expression of proinflammatory markers in white AT and plasma. A definitive role for immune cells in metabolic dysregulation was recently demonstrated in mice with myeloid cell-specific knock-out of IKKβ (9Arkan M.C. Hevener A.L. Greten F.R. Maeda S. Li Z.W. Long J.M. Wynshaw-Boris A. Poli G. Olefsky J. Karin M. Nat. Med. 2005; 11: 191-198Crossref PubMed Scopus (1505) Google Scholar), indicating that macrophage activation can cause systemic insulin resistance. Macrophages are a heterogeneous population of phagocytic cells found throughout the body that originate from the mononuclear phagocytic system (10Mantovani A. Sica A. Locati M. Immunity. 2005; 23: 344-346Abstract Full Text Full Text PDF PubMed Scopus (914) Google Scholar). These are highly plastic cells that arise from circulating myeloid-derived blood monocytes that have entered target tissues and gained the phenotypic and functional attributes of their tissue of residence. Like for other immune cells, the distribution and function of tissue macrophages have been largely characterized using monoclonal antibodies to cell surface proteins. In mice, the most commonly used monocyte/macrophage and myeloid cell surface markers are F4/80 and CD11b, although F4/80 and CD11b antibodies have been reported to react with eosinophils and dendritic cells and NK and other T and B cell subtypes, respectively (11Lai L. Alaverdi N. Maltais L. Morse H.C.R. J. Immunol. 1998; 160: 3861-3868PubMed Google Scholar). In AT, resident macrophages are surrounded by adipocytes that constantly release free fatty acids (FFAs) via lipolysis. FFAs thus have the potential to activate AT macrophages and consequently alter their function. FFAs can cause activation of JNK and IKKβ inflammatory pathways in adipose tissue, liver, and skeletal muscle (1Yuan M. Konstantopoulos N. Lee J. Hansen L. Li Z.W. Karin M. Shoelson S.E. Science. 2001; 293: 1673-1677Crossref PubMed Scopus (1652) Google Scholar, 2Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2679) Google Scholar, 12Wellen K.E. Hotamisligil G.S. J. Clin. Investig. 2005; 115: 1111-1119Crossref PubMed Scopus (3247) Google Scholar, 13Nguyen M.T. Satoh H. Favelyukis S. Babendure J.L. Imamura T. Sbodio J.I. Zalevsky J. Dahiyat B.I. Chi N.W. Olefsky J.M. J. Biol. Chem. 2005; 280: 35361-35371Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), leading to cellular inflammation and insulin resistance. In man, elevated FFA levels are a feature of obesity and type 2 diabetes (12Wellen K.E. Hotamisligil G.S. J. Clin. Investig. 2005; 115: 1111-1119Crossref PubMed Scopus (3247) Google Scholar, 14Boden G. Curr. Opin. Clin. Nutr. Metab. Care. 2002; 5: 545-549Crossref PubMed Scopus (180) Google Scholar). In animal models, high fat (HF) feeding and direct lipid infusion can increase plasma FFA levels and cause tissue and systemic inflammation and insulin resistance (15Kraegen E.W. Cooney G.J. Ye J.M. Thompson A.L. Furler S.M. Exp. Clin. Endocrinol. Diabetes. 2001; 109: S189-S201Crossref PubMed Scopus (117) Google Scholar, 16Boden G. Diabetes. 1997; 46: 3-10Crossref PubMed Scopus (0) Google Scholar). Toll-like receptors (TLRs) are thought to participate in sensing extracellular FFAs. TLRs belong to the Toll/interleukin-1 receptor superfamily and are widely expressed on cells of the immune system (17Takeda K. Akira S. Int. Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2752) Google Scholar, 18Liew F.Y. Xu D. Brint E.K. O'Neill L.A. Nat. Rev. Immunol. 2005; 5: 446-458Crossref PubMed Scopus (1272) Google Scholar). TLRs recognize bacteria-associated molecular patterns with high specificity, and TLR-mediated signal transduction leads to the activation of JNK and NFκB signaling pathways (17Takeda K. Akira S. Int. Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2752) Google Scholar, 19Guha M. Mackman N. Cell Signal. 2001; 13: 85-94Crossref PubMed Scopus (2005) Google Scholar), initiating the innate immune response. The saturated FA, lauric acid, was shown to activate TLR2 in 293T cells as well as NFκB-mediated, TLR4-dependent signaling pathways in RAW264.7 cells (20Lee J.Y. Zhao L. Youn H.S. Weatherill A.R. Tapping R. Feng L. Lee W.H. Fitzgerald K.A. Hwang D.H. J. Biol. Chem. 2004; 279: 16971-16979Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Similarly, an oleate/palmitate mixture can induce NFκB-dependent TLR4 signaling in 293T cells and cause inflammation in wild type (WT) but not TLR4-deficient adipocytes. The saturated FA palmitate was also shown to cause TLR4-dependent IκBα degradation in elicited peritoneal macrophages (21Shi H. Kokoeva M.V. Inouye K. Tzameli I. Yin H. Flier J.S. J. Clin. Investig. 2006; 116: 3015-3025Crossref PubMed Scopus (2768) Google Scholar). In the present study, we examined the effects of saturated and unsaturated FFAs in cultured RAW264.7 cells, primary bone marrow (BM)-derived cells and adipose tissue macrophages. We demonstrate that FFAs signal through both TLR2 and TLR4 to activate JNK and stimulate inflammatory pathways in CD11c+ myeloid cells. We also show that in AT, F4/80+CD11b+CD11c+ cells are the direct targets for FFAs and that HF feeding increases the number of these cells in AT. As such, these results provide a novel mechanistic link between lipid metabolism, inflammation, and insulin resistance. Reagents—Antibodies to IL-6, MCP-1,TNF-α, and horseradish peroxidase-linked secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and all other antibodies were from Cell Signaling (Beverly, MA). Tissue culture reagents were purchased from Invitrogen and Hyclone (Logan, UT). Cell Culture and Treatment—Mouse monocyte/macrophage RAW264.7 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's modified Eagle's medium (1g/liter glucose) supplemented with 10% low endotoxin FBS. In pretreated cells, 1 μm rosiglitazone or vehicle Me2SO was added at least 24 h prior to FFA treatment. RAW cells were then incubated for 3 h with either 500 μm of an FFA mixture containing equimolar amounts of tissue culture-grade arachidonic, lauric, linoleic, oleic, and myristic acids or with individual FFAs (500 μm) or ethanol vehicle in Dulbecco's modified Eagle's medium supplemented with FFA-free BSA (Sigma-Aldrich). All of the FFA solutions were pre-equilibrated with BSA at 37 °C for 1-1.5 h (22Spector A.A. John K. Fletcher J.E. J. Lipid Res. 1969; 10: 56-67Abstract Full Text PDF PubMed Google Scholar), and a 5:1 FFA:BSA ratio was used to simulate elevated FFAs levels. We ensured that all components for cell culture and treatment experiments contained very low or undetectable amounts of contaminating lipopolysaccharide by measuring endotoxin levels in the serum, culture medium, BSA preparation, and FFA stock solutions (LAL assay kit, Cambrex, East Rutherford, NJ). All of the solutions used contained undetectable amounts of endotoxin except for the BSA solution, which contained 0.3 EU/ml (0.03 ng/ml) of endotoxin. This amount of contaminating lipopolysaccharide was not sufficient to activate RAW264.7 cells and BMDCs. We found that treating RAW264.7 cells and BMDCs with 0.03 ng/ml of endotoxin for 3 h did not cause phosphorylation of JNK and secretion of TNF-α and JNK phosphorylation in these cells (data not shown). Western Blotting—The cells were lysed in cold buffer containing 50 mm HEPES, pH 7.4, 150 mm NaCl, 200 mm NaF, 20 mm sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 4 mm sodium orthovanadate, 2 mm phenylmethylsulfonyl fluoride, and 1 mm EDTA. Whole cell lysates (25 μg) or conditioned medium (30 μl) were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA). Following blocking with 5% milk in TBST, the membranes were probed with primary antibodies as indicated and subsequently incubated with horseradish peroxidase-linked secondary antibodies for chemiluminescent detection (Pierce). The blots were stripped in Restore™ Western blot stripping buffer (Pierce) and reprobed as necessary. Semi-quantitative RT-PCR—Total RNA was isolated and purified using RNeasy columns and RNase-free DNase according to the manufacturer's instructions (Qiagen). One-step RT-PCR kits (Qiagen) and sequence-specific primers (see supplemental Fig. S3) were used to generate RT-PCR products, which were then run on 8% acrylamide gels, stained with ethidium bromide, visualized, and quantitated using the Kodak ID Imaging station and software (Kodak Scientific Imaging Systems, New Haven, CT). Real Time PCR—Total RNA was isolated from cells as outlined above. First strand cDNA was synthesized using Super-Script III and random hexamers (Invitrogen). The samples were run in 20-μl reactions using an AB1 7300 (Applied Biosystems, Foster City, CA); SYBR Green oligonucleotides were used for detection, and sequence-specific primers are listed in supplemental Fig. S3. Gene expression levels were calculated after normalization to the standard housekeeping gene GAPDH using the ΔΔCT method as described by the manufacturer (Invitrogen) and expressed as relative mRNA levels compared with control. ELISA—Conditioned medium was assayed for mouse IL-1β, IL-6, MCP-1, and TNF-α using ELISA kits (BIOSOURCE, Camarillo, CA) following the manufacturer's protocol. Protein Knockdown—2 × 107 RAW264.7 cells were electroporated with 2.5 nmol of high pressure liquid chromatography-purified siRNA oligonucleotides (IDT, Coralville, IA) to mouse JNK1, JNK2, p65-NFκB, TLR2, or TLR4 or with luciferase control siRNA (oligonucleotide sequences in supplemental Fig. S3) using the XCell Gene Pulser (Bio-Rad). The cells were plated into 24-well tissue culture plates, and 24-48 h post-electroporation, they were treated with FFAs as described above. Conditioned medium and cell lysates were analyzed by immunoblotting. Isolation and Treatment of Bone Marrow-derived Cells—All of the animal experiments were performed humanely under protocols approved by the University of California, San Diego. TLR2/4 DKO male mice on a C57BL/6 background (kindly provided by Dr. Shizuo Akira, University of Osaka, Japan) and WT C57BL/6 male mice were maintained under specific pathogen-free conditions. Bone marrow cells were isolated from the femurs and tibias of 10-12-week-old homozygous and WT mice by flushing the medullary cavity with RPMI medium. After washing, the cells were seeded in tissue culture plates and differentiated into either bone marrow-derived macrophages (BMDMs) or bone marrow-derived dendritic cells (BMDCs) in RPMI medium containing 30% of L-929 conditioned medium or 10 or 40 ng/ml of recombinant GM-CSF, respectively, and supplemented with 20% low endotoxin FBS and streptomycin/penicillin. We observed that differentiating precursor cells in 40 ng/ml of GM-CSF yielded more BMDCs than when using 10 ng/ml of GM-CSF. However, this higher GM-CSF concentration did not affect BMDC proinflammatory gene expression (IL-6, TNF-α, IL-1β, and COX-2), differentiation and cell surface antigen expression (F4/80, CD11b, and CD11c), as determined by qPCR and FACS analysis (data not shown). We were also able to differentiate BMDMs from bone marrow precursor cells using murine recombinant M-CSF (data not shown). BMDM and BMDC differentiation was complete 8 days after cell plating; this was confirmed by the expression of F4/80, a marker preferentially expressed by mature macrophages, and of CD11c, a cell surface marker for dendritic cells, using FACS (data not shown). For FFA experiments, day 8 or 9 BMDMs and BMDCs were stimulated with 500 μm FFA for 3 h, and conditioned medium and cell lysates were analyzed as indicated. Immunohistocytochemistry—Paraffin-embedded adipose tissue was sectioned, deparaffinized, and rehydrated prior to endogenous peroxidase and biotin removal using 0.03% H2O2 and 0.1% avidin. The slides were heated in 0.1 m citrate buffer for antigen retrieval, cooled, washed, and then blocked with 1% BSA in TBST. The sections were incubated overnight with CD11c primary antibody (eBioscience, San Diego, CA). Biotin-conjugated secondary and horseradish peroxidase-conjugated streptavidin tertiary antibodies were applied for detection, and the sections were developed in substrate chromogen, counterstained in Mayers Hematoxylin, and mounted in Gelmount Biomedia. Frozen quad muscle tissue was sectioned (5 um), air-dried, and fixed in acetone. After endogenous peroxidase removal using 0.03% H2O2, the sections were washed in PBS and blocked in 0.01% biotin for 15 min and then in 1% BSA in PBS for 30 min. Biotin-conjugated CD11c antibody was overlaid for 1 h, and the sections were further processed as outlined above. Animal Studies—Wild type male C57BL/6 and ob/obJ male mice were purchased from Harlan (Indianapolis, IN). The animals were housed in a pathogen-free facility with a 12-h light/12-h dark cycle and given free access to food and water. C57BL/6 mice were placed on a HFD consisting of 40% of calories from fat (TD.96132; Harlan Teklad, Madison, WI) starting at 12 weeks of age for 1, 12, or 20 weeks. Control C57BL/6 mice were fed a standard diet with 12% of calories from fat (LabDiet, 5001; Richmond, IN). The body weights and food intakes were recorded every week (data not shown). An insulin tolerance test (ITT) was performed on mice after 1 and 12 weeks of chow or HFD feeding. The mice were fasted for 6 h. After 4 h of fasting, basal plasma glucose was measured. At 6 h, the mice were injected intraperitoneal with 0.6 units of insulin/kg of body weight. Blood glucose was measured through the tail tip at the indicated times, using a OneTouch glucose-monitoring system (Lifescan, Milpitas, CA). Stromal Vascular Fraction (SVF) Isolation and FACS Analysis—Epididymal fat pads were excised from male C57BL/6 mice fed normal chow (NC) or HFD, weighed, rinsed three times in PBS, and then minced in FACS buffer (PBS + 1% low endotoxin BSA). Tissue suspensions were centrifuged at 500 × g for 5 min and then collagenase-treated (1 mg/ml; Sigma-Aldrich) for 30 min at 37 °C with shaking. The cell suspensions were filtered through a 100-μm filter and centrifuged at 500 × g for 5 min. SVF pellets were then incubated with RBC lysis buffer (eBioscience) for 5 min prior to centrifugation (300 × g for 5 min) and resuspension in FACS buffer. Stromal vascular cells (SVCs) were incubated with Fc Block (BD Biosciences, San Jose, CA) for 20 min at 4 °C prior to staining with fluorescently labeled primary antibodies or control IgGs for 25 min at 4 °C. F4/80-allophycocyanin FACS antibody was purchased from AbD Serotec (Raleigh, NC); all other fluorescein isothiocyanate- and phycoerythrin-conjugated FACS antibodies were from BD Biosciences. The cells were gently washed twice and resuspended in FACS buffer with propidium iodide (Sigma-Aldrich). SVCs were analyzed using FACSCalibur and FACSAria flow cytometers (BD Biosciences). Unstained, single stains, and fluorescence minus one controls were used for setting compensation and gates. Forcellsorting,F4/80+CD11b+CD11c−andF4/80+CD11b+-CD11c+ cells from lean, NC SVFs were sorted into FBS. Sorted cells were allowed to recover for 2 h. The cells were then washed and treated with FFA for subsequent RNA extraction and real time PCR analysis. Glucose Uptake Assay—BMDCs from WT and TLR2/4 DKO mice were treated with 500 μm FFA or vehicle for 3 h and then washed twice with RPMI medium to remove the FFAs. The cells were subsequently incubated with fresh culture medium containing 2% FBS. After 6 h of incubation, CM was harvested for ELISA analysis and co-culture experiments. L6 myocytes were cultured in α-minimal essential medium supplemented with 10% FBS and differentiated into myotubes in α-minimal essential medium containing 2% FBS for ∼6-7 days. The L6 myotube culture medium was then replaced with CM from BMDCs diluted 1:1 in fresh L6 differentiation medium. L6 myotubes were incubated with BMDC-derived CM for 24-48 h prior to assaying glucose uptake. For glucose uptake assays, L6 myotubes were serum starved for 3 h in α-minimal essential medium with 0.5% FFA-free BSA and then glucose-starved for 30 min in HEPES salt buffer containing 0.5% FFA-free BSA. The cells were stimulated with insulin (5 nm) at 37 °C for 20 min; tracer glucose was then added for 10 min. After 30 min of insulin stimulation, glucose uptake was assayed in quadruplicate wells for each condition using 1,2-3H-2-deoxy-d-glucose (0.2 μCi, 0.1 mm, 10 min) in four independent experiments. Data Analysis—Densitometric quantification and normalization were performed using the NIH Image 1.63 software. The values presented are expressed as the means ± S.E. The statistical significance of the differences between various treatments was determined by one-way analysis of variance with the Bonferroni correction. FFAs Cause a Proinflammatory Response in RAW264.7 Cells—Recent studies suggest that chronic inflammation in AT is an important mechanism underlying the insulin resistance associated with obesity, HF feeding, and diabetes and that infiltrating macrophages may be responsible for the activation of proinflammatory pathways (6Bouloumie A. Curat C.A. Sengenes C. Lolmede K. Miranville A. Busse R. Curr. Opin. Clin. Nutr. Metab. Care. 2005; 8: 347-354Crossref PubMed Scopus (228) Google Scholar, 23Kolb H. Mandrup-Poulsen T. Diabetologia. 2005; 48: 1038-1050Crossref PubMed Scopus (345) Google Scholar). Because FFAs are released in AT, we studied the effects of FFAs on AT macrophage function. We first treated RAW264.7 murine monocyte/macrophage cells with a mixture of saturated and unsaturated FFAs and surveyed various stress/inflammatory signaling responses. FFA treatment broadly activated the JNK and IKKβ signaling pathways (Fig. 1A), in a time- and concentration-dependent manner, with JNK activation being observed as early as 5 min after FFA treatment (data not shown). After washing to remove FFAs, followed by a 12-h recovery period, inflammatory markers returned to base-line levels, and the cells were once again responsive to lipopolysaccharide stimulation (data not shown). Thus, the proinflammatory effects of FFAs were specific and reversible and did not result from cell toxicity. When the cells were treated with 500 μm FFA for 3 h, we observed induction of the proinflammatory genes IL-1β, IL-6, MCP-1, and MMP-9, an increase in intracellular MCP-1 and TNF-α levels, and an increase in secreted proinflammatory chemo/cytokines IL-1β, IL-6, MCP-1, and TNF-α (supplemental Fig. S1, A-C). When we tested individual FAs, each FA in the mixture, except for myristic acid, was able to activate the proinflammatory kinase pathways in RAW264.7 cells and increased intracellular and secreted TNF-α levels, albeit to different degrees (supplemental Fig. S1, D and E). Overall, the unsaturated FA arachidonic acid was most potent in causing these effects. We assessed whether a TZD, rosiglitazone, could inhibit FFA-induced inflammation in RAW264.7 cells and found that all the FFA-induced effects were attenuated by pretreatment with rosiglitazone (supplemental Fig. S2, A-C). Contribution of JNK and IKKβ-NFκB to the FFA Proinflammatory Effects—Previous reports have documented that FFAs induce mostly NFκB-dependent effects, and we observed that FFA treatment activated both JNK and IKKβ signaling pathways. Therefore, we assessed the contribution of these two kinases to the overall FFA effect using siRNAs targeted specifically against JNK1, JNK2, and p65-NFκB. JNK1 and JNK2 siRNAs decreased levels of both JNK protein isoforms by >70% (13Nguyen M.T. Satoh H. Favelyukis S. Babendure J.L. Imamura T. Sbodio J.I. Zalevsky J. Dahiyat B.I. Chi N.W. Olefsky J.M. J. Biol. Chem. 2005; 280: 35361-35371Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), and p65-NFκB siRNA decreased p65 protein levels by ∼75% (Fig. 1B). JNK knockdown (KD) significantly reduced basal MMP-9, IL-1β, TNF-α, and COX-2 gene expression levels, and inhibited the FFA-induced up-regulation in IL-1β and MMP-9 gene expression (Fig. 1C). In contrast, knocking down p65 produced smaller inhibitory effects on gene expression. Moreover, JNK KD resulted in decreased FFA-induced intracellular and secreted TNF-α levels (Fig. 1D), whereas p65 KD, while having the expected effect to decrease phosphorylation of NFκB, had little or no effect on intracellular and secreted TNF-α (Fig. 1E). This suggested that JNK plays a more central role in mediating the FFA-induced proinflammatory effects in RAW264.7 cells. We thus focused on examining the role of JNK kinase in mediating the effects of FFAs. TLR2 and TLR4 Mediate FA Signaling in RAW264.7 Cells—TLRs are expressed on monocytes and macrophages, and it has been reported that bacterial lipopolysaccharide, which contains an FA moiety, can induce proinflammatory effects through TLR4 (17Takeda K. Akira S. Int. Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2752) Google Scholar, 24Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6514) Google Scholar, 25Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar). Thus, we hypothesized that TLR4, and possibly TLR2, another member of the TLR family, could mediate the FFA proinflammatory signals. To test this idea, siRNAs were used to knockdown TLR4 and TLR2 proteins in RAW264.7 cells (Fig. 2A), achieving ∼50% and ∼75% KD efficiency for TLR2 and TLR4, respectively (bar graph in Fig. 2A). TLR2 KD significantly reduced basal expression of MMP-9, IL-1β, and TNF-α genes, whereas TLR4 KD reduced the basal expression of these genes and that of COX-2 (Fig. 2B). TLR2 and TLR4 KD also inhibited the FFA-induced up-regulation in IL-1β and MMP-9 gene expression. Fig. 2C shows that in the TLR2 KD cells, FFA-induced JNK phosphorylation was inhibited, as was the increase in intracellular and secreted TNF-α levels. When TLR4 was depleted, these proinflammatory responses were also inhibited, but to a greater extent (Fig. 2D). These results indicate that both TLR4 and TLR2 play a role in mediating FA signaling via JNK activation. Studies in Bone Marrow-derived Cells—RAW264.7 cells have been extensively used as a model cell line for studies of macrophage biology. We found that RAW264.7 cells express high levels of the macrophage and myeloid cell surface markers F4/80 and CD11b but also high levels of a cell surface marker more typical of dendritic and T cells, CD11c (data not shown). With this in mind, and given the heterogeneity and plasticity of the macrophage population, we further examined the effects of FFAs in two types of primary myeloid-derived cell types: BMDMs, which express F4/80, CD11b but not CD11c, and BMDCs, which express all three markers. BMDMs and BMDCs were prepared as described in detail under “Experimental Procedures.” According to previously established methods, the phenotype of these primary cells was confirmed by FACS using fluorescently labeled antibodies showing that BMDMs express F4/80 and CD11b, whereas BMDCs are positive for F4/80, CD11b, and CD11c (data not shown). To characterize these two cell types, we examined the expression of inflammatory genes by qPCR (Fig. 3A). The relative expression of COX-2, CCR2, IL-1β, IL-6, and TNF-α genes was strikingly higher in BMDCs than BMDMs (p < 0.05 or less), suggesting that BMDCs possess greater proinflammatory activity than BMDMs. Consistent with this, BMDMs expressed high levels of anti-inflammatory cytokine IL-10, whereas BMDCs exhibited no detectable levels of IL-10. BMDMs and BMDCs have been shown to express TLR2 and TLR4. Because BMDCs have heightened proinflammatory activity and because FFAs exert their inflammat
Insulin-stimulated kinase activity of adipocyte-derived insulin receptors is reduced in subjects with non-insulin-dependent diabetes mellitus (NIDDM) but normal in obese nondiabetics. To assess the reversibility of the kinase defect in NIDDM, insulin receptor kinase activity was measured before and after weight loss in 10 NIDDM and 5 obese nondiabetic subjects. Peripheral insulin action was also assessed in vivo by glucose disposal rates (GDR) measured during a hyperinsulinemic (300 mU/M2 per min) euglycemic clamp. In the NIDDMs, insulin receptor kinase activity was reduced by 50-80% and rose to approximately 65-90% (P less than 0.01) of normal after 13.2 +/- 2.0 kg (P less than 0.01) weight loss; comparable weight loss (18.2 +/- 1.5 kg, P less than 0.01) in the nondiabetics resulted in no significant change in insulin receptor kinase activity. Relative to GDR measured in lean nondiabetics, GDR in the NIDDMs was 35% of normal initially and 67% (P less than 0.01) of normal after diet therapy; weight loss in the nondiabetics resulted in an increase in GDR from 53 to 76% of normal (P less than 0.05). These results indicate that the insulin receptor kinase defect that is present in NIDDM is largely reversible after weight reduction. In contrast, the improvement in GDR, in the absence of any change in insulin receptor kinase activity in the nondiabetics, suggests that the main cause of insulin resistance in obesity lies distal to the kinase.
To assess the role of insulin receptor (IR) tyrosine kinase in human insulin resistance, we examined the kinase activity of IR of skeletal muscle biopsies from eight lean and five obese nondiabetics and six obese subjects with noninsulin-dependent diabetes mellitus (NIDDM). Biopsies were taken during euglycemic clamps at insulin infusion rates of 0, 40, 120, and 1200 mU/m2.min. IRs were immobilized on insulin agarose beads, and autophosphorylation and histone 2B phosphorylation were measured. Phosphatase and protease inhibitors preserved the in vivo phosphorylation state of the IRs. Glucose disposal rates (GDR) were reduced according to insulin dose by 23-30% in the obese (P < 0.05) and 43-64% in the NIDDM subjects (P < 0.0005). IR autophosphorylation was increased up to 9-fold in controls and was reduced (P = 0.04) in NIDDM compared to obese subjects. Histone-2B kinase was increased up to 6-fold in controls and was reduced by 50% in NIDDM. Kinase values by both methods were similar in lean and obese controls. In vivo stimulation of kinase was well correlated to the increase in GDR, as was the decrement in kinase in NIDDM to the decrement in GDR. These results suggest that defects in muscle IR kinase are significant in the in vivo insulin resistance of NIDDM, but not that of obesity.
Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Comprehensive genome-wide analysis of the epigenetic and transcription status of the TLR4-induced transcriptional program in macrophages suggests that Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early- (I/E) and late-response genes results from a sequential process in which signal-independent factors initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by encoding a distinct set of signal-dependent transcription factor elements, including TATA boxes, which lead to preferential binding of TBP and basal enrichment for RNA polymerase II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases the overall rates of both transcriptional initiation and the efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. Collectively, these findings reveal broadly utilized mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses.
The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-d-manno-octulosonic acid (Kdo)2-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo2-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and 1H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo2-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >103 in cells from TLR-4-deficient mice. The purity of Kdo2-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2. The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-d-manno-octulosonic acid (Kdo)2-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo2-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and 1H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo2-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >103 in cells from TLR-4-deficient mice. The purity of Kdo2-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2. The LIPID MAPS consortium is developing quantitative methods for evaluating the composition, biosynthesis, and function of all macrophage lipids (1Dennis E.A. Brown H.A. Deems R. Glass C.K. Merrill A.H. Murphy R.C. Raetz C.R.H. Shaw W. Subramaniam S. Russell D.W. et al.The LIPID MAPS approach to lipidomics.in: Feng L. Prestwich G.D. Functional Lipidomics. CRC Press/Taylor and Francis Group, Boca Raton, FL2005: 1-15Crossref Google Scholar). These amphipathic substances not only are structural components of biological membranes but also play important roles in the pathophysiology of inflammation, atherosclerosis, and growth control. Additional lipid functions should emerge from the comprehensive analysis of macrophage lipids. Electrospray ionization/mass spectrometry (ESI/MS) (2Murphy R.C. Fiedler J. Hevko J. Analysis of nonvolatile lipids by mass spectrometry.Chem. Rev. 2001; 101: 479-526Crossref PubMed Scopus (235) Google Scholar, 3Pulfer M. Murphy R.C. Electrospray mass spectrometry of phospholipids.Mass Spectrom. Rev. 2003; 22: 332-364Crossref PubMed Scopus (733) Google Scholar), coupled with prefractionation methods like reverse-phase liquid chromatography (LC), is being applied systematically to set the stage for the seamless integration of lipid metabolism into the broader fields of genomics, proteomics, and systems biology. To facilitate this endeavor, LIPID MAPS has introduced a new comprehensive classification system for biological lipids, amenable to computer-based data processing and substructure comparison (4Fahy E. Subramaniam S. Brown H.A. Glass C.K. Merrill Jr., A.H. Murphy R.C. Raetz C.R.H. Russell D.W. Seyama Y. Shaw W. et al.A comprehensive classification system for lipids.J. Lipid Res. 2005; 46: 839-862Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar). The eight LIPID MAPS categories are 1) fatty acyls, 2) glycerolipids, 3) glycerophospholipids, 4) sphingolipids, 5) sterol lipids, 6) prenol lipids, 7) saccharolipids, and 8) polyketides. More details are available on the LIPID MAPS web site (www.lipidmaps.org). As an initial test of the LIPID MAPS approach, the time-dependent response of the macrophage to stimulation by lipopolysaccharide (LPS) is being investigated. LPS is a potent activator of the innate immunity receptor TLR-4/MD2 (5Aderem A. Ulevitch R.J. Toll-like receptors in the induction of the innate immune response.Nature. 2000; 406: 782-787Crossref PubMed Scopus (2624) Google Scholar, 6Raetz C.R.H. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar, 7Gangloff M. Gay N.J. MD-2: the Toll ‘gatekeeper’ in endotoxin signalling.Trends Biochem. Sci. 2004; 29: 294-300Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). LPS, a saccharolipid glycan according to the new LIPID MAPS classification scheme (4Fahy E. Subramaniam S. Brown H.A. Glass C.K. Merrill Jr., A.H. Murphy R.C. Raetz C.R.H. Russell D.W. Seyama Y. Shaw W. et al.A comprehensive classification system for lipids.J. Lipid Res. 2005; 46: 839-862Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar), is present in the outer membranes of most Gram-negative bacteria (6Raetz C.R.H. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar, 8Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zaähringer U. Seydel U. Di Padova F. et al.Bacterial endotoxin: molecular relationships of structure to activity and function.FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1326) Google Scholar, 9Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999Google Scholar, 10Nikaido H. Molecular basis of bacterial outer membrane permeability revisited.Microbiol. Mol. Biol. Rev. 2003; 67: 593-656Crossref PubMed Scopus (2869) Google Scholar). It stimulates macrophages via its lipid anchor, which is termed lipid A (or endotoxin). Animal cells can detect picomolar lipid A concentrations using TLR-4/MD2 and accessory proteins such as CD14 and LPS binding protein (5Aderem A. Ulevitch R.J. Toll-like receptors in the induction of the innate immune response.Nature. 2000; 406: 782-787Crossref PubMed Scopus (2624) Google Scholar, 7Gangloff M. Gay N.J. MD-2: the Toll ‘gatekeeper’ in endotoxin signalling.Trends Biochem. Sci. 2004; 29: 294-300Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 11Ulevitch R.J. Therapeutics targeting the innate immune system.Nat. Rev. Immunol. 2004; 4: 512-520Crossref PubMed Scopus (267) Google Scholar, 12Visintin A. Halmen K.A. Latz E. Monks B.G. Golenbock D.T. Pharmacological inhibition of endotoxin responses is achieved by targeting the TLR4 coreceptor, MD-2.J. Immunol. 2005; 175: 6465-6472Crossref PubMed Scopus (130) Google Scholar). Human volunteers injected with 4 ng/kg of LPS develop fever and a flu-like illness, lasting 24 to 48 h (13Lynn M. Rossignol D.P. Wheeler J.L. Kao R.J. Perdomo C.A. Noveck R. Vargas R. D'Angelo T. Gotzkowsky S. McMahon F.G. Blocking of responses to endotoxin by E5564 in healthy volunteers with experimental endotoxemia.J. Infect. Dis. 2003; 187: 631-639Crossref PubMed Scopus (141) Google Scholar). Recent clinical studies in humans have characterized the complex response to endotoxin injection by using micro-array technology (14Prabhakar U. Conway T.M. Murdock P. Mooney J.L. Clark S. Hedge P. Bond B.C. Jazwinska E.C. Barnes M.R. Tobin F. et al.Correlation of protein and gene expression profiles of inflammatory proteins after endotoxin challenge in human subjects.DNA Cell Biol. 2005; 24: 410-431Crossref PubMed Scopus (46) Google Scholar, 15Calvano S.E. Xiao W. Richards D.R. Felciano R.M. Baker H.V. Cho R.J. Chen R.O. Brownstein B.H. Cobb J.P. Tschoeke S.K. et al.A network-based analysis of systemic inflammation in humans.Nature. 2005; 437: 1032-1037Crossref PubMed Scopus (1219) Google Scholar). This approach revealed that the transcription of hundreds of genes is activated or repressed in human leukocytes following exposure to endotoxin (15Calvano S.E. Xiao W. Richards D.R. Felciano R.M. Baker H.V. Cho R.J. Chen R.O. Brownstein B.H. Cobb J.P. Tschoeke S.K. et al.A network-based analysis of systemic inflammation in humans.Nature. 2005; 437: 1032-1037Crossref PubMed Scopus (1219) Google Scholar). Excessive endotoxin exposure during severe Gram-negative sepsis contributes to shock, multiple organ failure, and death. A promising approach to the amelioration of endotoxin-induced illnesses has emerged with the discovery that certain synthetic lipid A analogs (13Lynn M. Rossignol D.P. Wheeler J.L. Kao R.J. Perdomo C.A. Noveck R. Vargas R. D'Angelo T. Gotzkowsky S. McMahon F.G. Blocking of responses to endotoxin by E5564 in healthy volunteers with experimental endotoxemia.J. Infect. Dis. 2003; 187: 631-639Crossref PubMed Scopus (141) Google Scholar, 16Hawkins L.D. Christ W.J. Rossignol D.P. Inhibition of endotoxin response by synthetic TLR4 antagonists.Curr. Top. Med. Chem. 2004; 4: 1147-1171Crossref PubMed Scopus (90) Google Scholar) or precursors (17Golenbock D.T. Hampton R.Y. Qureshi N. Takayama K. Raetz C.R.H. Lipid A-like molecules that antagonize the effects of endotoxins on human monocytes.J. Biol. Chem. 1991; 266: 19490-19498Abstract Full Text PDF PubMed Google Scholar) can antagonize the effects of lipid A (endotoxin) on TLR-4/MD2. The former are currently in clinical trials (13Lynn M. Rossignol D.P. Wheeler J.L. Kao R.J. Perdomo C.A. Noveck R. Vargas R. D'Angelo T. Gotzkowsky S. McMahon F.G. Blocking of responses to endotoxin by E5564 in healthy volunteers with experimental endotoxemia.J. Infect. Dis. 2003; 187: 631-639Crossref PubMed Scopus (141) Google Scholar, 16Hawkins L.D. Christ W.J. Rossignol D.P. Inhibition of endotoxin response by synthetic TLR4 antagonists.Curr. Top. Med. Chem. 2004; 4: 1147-1171Crossref PubMed Scopus (90) Google Scholar). A limitation of using native LPS from wild-type Gram-negative bacteria for clinical or biological studies of endotoxin activity is its large size and micro-heterogeneity, especially in the length and composition of its terminal glycan chains (6Raetz C.R.H. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar, 9Brade H. Opal S.M. Vogel S.N. Morrison D.C. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1999Google Scholar). Direct detection and quantification by mass spectrometry of intact LPS in blood or biological samples is not yet possible. Accordingly, LPS levels are usually estimated by indirect methods, such as the clotting of the amoebocyte limulus lysate induced by the lipid A moiety of LPS (18Bryans T.D. Braithwaite C. Broad J. Cooper J.F. Darnell K.R. Hitchins V.M. Karren A.J. Lee P.S. Bacterial endotoxin testing: a report on the methods, background, data, and regulatory history of extraction recovery efficiency.Biomed. Instrum. Technol. 2004; 38: 73-78Crossref PubMed Scopus (24) Google Scholar, 19Cohen J. The detection and interpretation of endotoxaemia.Intensive Care Med. 2000; 26: 51-56Crossref PubMed Google Scholar). The tissue distribution and metabolism of LPS injected into animals have likewise been difficult to evaluate because of the same micro-heterogeneity problem. Knowledge of endotoxin tissue levels and metabolism might suggest new therapeutic approaches to problems of sepsis and inflammation. We now report the large-scale purification, structural analysis, and biological characterization of a chemically defined LPS, consisting of lipid A and an attached 3-deoxy-d-manno-octulosonic acid (Kdo) disaccharide (Fig. 1, compound A) (6Raetz C.R.H. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). This substance, designated Kdo2-Lipid A, was purified from an Escherichia coli K-12 mutant that synthesizes a truncated LPS because of a mutation in the enzymes that normally attach the heptose residues of the LPS core domain (6Raetz C.R.H. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar, 20Brabetz W. Muller-Loennies S. Holst O. Brade H. Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution.Eur. J. Biochem. 1997; 247: 716-724Crossref PubMed Scopus (107) Google Scholar, 21Murphy R.C. Raetz C.R.H. Reynolds C.M. Barkley R.M. Mass spectrometry advances in lipidomica: collision-induced decomposition of Kdo(2)-lipid A.Prostaglandins Other Lipid Mediat. 2005; 77: 131-140Crossref PubMed Scopus (23) Google Scholar). This LPS preparation has an intact lipid A anchor, which is fully active as an endotoxin by various biological criteria, such as the stimulation of RAW 264.7 macrophage-like tumor cells to produce eicosanoids and tumor necrosis factor-α (TNFα). Kdo2-Lipid A is highly selective for TLR-4 and has the distinct advantage that it can be quantified by ESI/MS. The chemical purity of Kdo2-Lipid A is sufficient to enable high-resolution structural studies, such as NMR spectroscopy or X-ray crystallography of its complexes with important receptor proteins or enzymes (7Gangloff M. Gay N.J. MD-2: the Toll ‘gatekeeper’ in endotoxin signalling.Trends Biochem. Sci. 2004; 29: 294-300Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Tryptone, yeast extract, L-broth, tetracycline, and glucose were obtained from Difco. Chloroform and methanol were purchased from EMS. Cyclohexane was obtained from Burdick and Jackson. EDTA, t-butanol, and t-butyl ammonium phosphate (TBAP) were from Aldrich. Ammonium acetate and acetonitrile were obtained from Acros. Ethanol, ammonium hydroxide, isopropanol, and sodium chloride were from Fisher Scientific (Fair Lawn, NJ). Silica gel was provided by Grace Davison. Silica gel modified by Astec was used as the C18 reverse-phase resin. Whatman DE 23 DEAE cellulose ion exchange resin and Whatman Partsil K6 TLC plates were purchased from VWR. Lipid visualization after TLC was performed by spraying the plates with 10% H2SO4 in ethanol, followed by charring. RAW 264.7 cells were obtained from the American Type Culture Collection. They were grown for no more than 24 passages on DMEM (Cellgro), supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Invitrogen; Carlsbad, CA). For cell stimulation experiments, the LPS from E. coli 0111:B4 was obtained from Sigma (# L4130). Kdo2-Lipid A was prepared as described below. All liquid chromatography/mass spectrometry (LC/MS) solvents used for prostaglandin analysis were OmniSolv grade or the equivalent from EMD Chemicals (Gibbstown, NJ). The formic acid was purchased from Fisher Scientific. All other fine chemicals were analytical reagent grade or better. The heptose-deficient E. coli mutant WBB06 (20Brabetz W. Muller-Loennies S. Holst O. Brade H. Deletion of the heptosyltransferase genes rfaC and rfaF in Escherichia coli K-12 results in an Re-type lipopolysaccharide with a high degree of 2-aminoethanol phosphate substitution.Eur. J. Biochem. 1997; 247: 716-724Crossref PubMed Scopus (107) Google Scholar) was grown on a modified Luria-Bertani broth (22Kanipes M.I. Lin S. Cotter R.J. Raetz C.R.H. Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.J. Biol. Chem. 2001; 276: 1156-1163Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). A 500 l capacity IF-500 fermentor was filled with 375 l of water, containing 3,750 g of tryptone (10 g/l), 1,875 g yeast extract (5 g/l), 3,750 g NaCl (10 g/l), and 750 g glucose (2 g/l). After sterilization, 4.5 g of tetracycline (12 mg/l) was added from a concentrated filter-sterilized stock solution in 70% aqueous ethanol. The pH of the growth medium was adjusted to 7.2 with NaOH and H3PO4. After 24 h of growth to early stationary phase at 37°C, the WBB06 cells were harvested by centrifugation in a CEPA Z-41 continuous-flow centrifuge. The cell paste (approximately 2 kg) was frozen at −80°C. The overall scheme for the purification is summarized in Fig. 2. A modified Folch extraction procedure was used to recover the lipids (23Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). The frozen WBB06 cells were dispersed in a single-phase solvent mixture consisting of 15 ml chloroform-methanol (2:1; v/v) per gram of cell paste. The suspended paste was stirred overnight at room temperature. The insoluble residue was removed, and the supernatant, containing the extracted lipids, was stored at −20°C. The residue was reextracted overnight at room temperature with 7.5 ml of chloroform-methanol (2:1; v/v) per gram of initial cell paste. The reextracted residue was discarded, and the second supernatant was pooled with the first. The combined supernatants were converted to a two-phase system by adding 20% of their total volume as aqueous 1.0 M NaCl. The lower organic phase was recovered, and the solvent was removed by rotary evaporation. The weight of the extracted lipids, which consist mainly of phosphatidyl-ethanolamine, phosphatidylglycerol, and Kdo2-Lipid A, was approximately 65.3 g, or 3.2% of the initial cell paste weight. The extracted lipids were stored neat at −20°C. The Kdo2-Lipid A was purified from the WBB06 membrane phospholipids on a normal-phase silica column, consisting of 20 g silica gel per gram total lipid. The silica was prepared by washing with two column volumes of 2% ammonium EDTA in methanol-water (1:1; v/v), followed by extensive washing with methanol-water (1:1; v/v) until the eluant pH was less than 8. The column was then washed with methanol, followed by chloroform. The extracted lipids were dissolved and loaded onto the column in chloroform-methanol (8:2; v/v). The lipids were eluted with a gradient that started at chloroform-methanol-water, 80:20:2 (v/v/v) and ended at 65:35:8 (v/v/v). Fractions containing Kdo2-Lipid A were identified by TLC (Rf, ∼0.3) using the mobile-phase chloroform-methanol-water (65:35:8; v/v/v). The fractions were pooled, and solvent was removed by rotary evaporation. The weight of lipids in the pooled fractions was 9.0 g, or 15% of the total lipid weight loaded onto the column. The Kdo2-Lipid A was ∼70% pure at this stage, as judged by HPLC (see below). Purification by reverse-phase chromatography was performed as described previously on a C18 column to separate the predominant hexa-acylated Kdo2-Lipid A (Fig. 1) from minor hepta-, penta- and tetra-acylated lipid A species, and from minor phospholipid contaminants, using a two-solvent mixture. Solvent A is acetonitrile-water (1:1) with 1 mM TBAP, and solvent B consists of isopropanol-water (85:15; v/v) with 1 mM TBAP in water (22Kanipes M.I. Lin S. Cotter R.J. Raetz C.R.H. Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.J. Biol. Chem. 2001; 276: 1156-1163Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The reverse-phase chromatography column consisted of 60 g of resin per gram of lipid. The pooled fractions, containing the predominant hexa-acylated form of Kdo2-Lipid A, were extracted with chloroform and aqueous 0.5 M ammonium acetate. Solvent was removed by rotary evaporation, and the lipid was stored neat at −20°C. Removal of TBAP and remaining pigments was accomplished by column chromatography on DEAE-cellulose in the solvent chloroform-methanol-water (2:3:1; v/v/v) with ammonium acetate elution (22Kanipes M.I. Lin S. Cotter R.J. Raetz C.R.H. Ca2+-induced phosphoethanolamine transfer to the outer 3-deoxy-D-manno-octulosonic acid moiety of Escherichia coli lipopolysaccharide. A novel membrane enzyme dependent upon phosphatidylethanolamine.J. Biol. Chem. 2001; 276: 1156-1163Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). The DEAE-cellulose column consisted of 30 ml DEAE cellulose bed volume per gram of lipid. Pooled fractions containing the Kdo2-Lipid A were extracted with chloroform and aqueous 0.5 M ammonium acetate. Solvent was removed by rotary evaporation, and the lipids were stored neat at −20°C. Final purification to remove residual contaminants was achieved with a second normal-phase chromatography column, consisting of 20 g of silica gel per gram of lipid. Kdo2-Lipid A dissolved in chloroform-methanol was applied to the column, which was eluted as described above. Solvent was removed by rotary evaporation from the final pooled fractions containing the Kdo2-Lipid A. The residue was reconstituted in cyclohexane-t-butanol (1:1;v/v) and freeze dried. The final overall yield obtained by this procedure was 1.4 g of hexa-acylated Kdo2-Lipid A as the ammonium salt (>95% pure by HPLC). Long-term storage at −16°C to −24°C is recommended. The Kdo2-Lipid A in the ampoules was prepared for use in cell stimulation experiments by dispersing it in sterile Dulbecco’s phosphate-buffered saline (DPBS) at 1 mg/ml by sonic irradiation in a bath sonicator for 5 min. A 100 μl portion of this stock was then diluted with 900 μl sterile DPBS. This 100 μg/ml (1,000×) aqueous dispersion of Kdo2-Lipid A was subjected to a second sonic irradiation prior to being added to the cells to yield a final concentration of 100 ng/ml in the growth medium. HPLC analysis of Kdo2-Lipid A was performed using an Agilent 1100 quaternary pump system, equipped with a temperature-controlled column compartment and an in-line solvent degassing unit. The Zorbax Eclipse XDB-C8 column (150 mm × 4.6 mm, 5 μ particle size) (Agilent Technologies; Palo Alto, CA) was maintained at 40°C. Samples were eluted using a gradient that consisted of methanol-water-chloroform (62:36:2; v/v/v) with 10 mM ammonium acetate as mobile-phase A and chloroform-methanol-water (80:20:2; v/v/v) with 50 mM ammonium acetate as mobile-phase B. The flow rate was 1 ml/min. Chemicals and solvents were HPLC grade and purchased from Fisher Scientific. The initial solvent, consisting of 85% A and 15% B, was maintained for 2 min, followed by a linear gradient to a final composition of 70% A and 30% B after 20 min; the solvent was held at the same composition until 30 min. A 10 min reequilibration of the column with 85% A and 15% B was performed prior to the next injection. The material eluting from the column was directed to a SEDEX model 75C evaporative light-scattering detector (S.E.D.E.R.E., France). Zero-grade compressed air was used to nebulize the postcolumn flow stream at 3.5 bar into the detector at 50°C, set at a photomultiplier gain of 6. The detector signal was transferred to the Agilent HPLC Chemstation software for integration. The Kdo2-Lipid A samples were prepared at 1 mg/ml in a mixture of 85% A and 15% B (as defined above). Ten micrograms was injected onto the column for purity assessment by HPLC and evaporative light-scattering detection (ELSD). Flow injection analysis of Kdo2-Lipid A was carried out using an ABI 4000 Q Trap® tandem quadrupole mass spectrometer (Applied Biosystems; Thornhill, Ontario, Canada) with negative-ion electrospray ionization during infusion of 10 μl/min of acetonitrile-water (70:30; v/v) containing 10 mM of ammonium acetate. The electrospray voltage was set at −4.5 kV, and nitrogen was used as curtain gas. LC/MS and low-resolution LC/MS/MS were carried out using a reverse-phase C8 HPLC column (Agilent), operated at a flow rate of 1 ml/min under gradient conditions, as described above. At this flow rate, the turbo ion spray interface was set at 600°C. For MS/MS experiments, nitrogen was used as collision gas at a collision energy of −15 V (laboratory frame of reference). High-resolution electrospray mass spectrometry was carried out using a tandem hybrid quadrupole time-of-flight mass spectrometer (ABI Qstar XL, Applied Biosystems) by infusion of 50 ng/μl Kdo2-Lipid A in chloroform-methanol (2:1; v/v) at 6 μl/min. The instrument was scanned from m/z 100–2,500 in a negative-ion mode with the electrospray set at −4.2 kV. Nitrogen was used as collision gas and collisional activation of ions was performed at −40.0 V (laboratory frame of reference). This instrument was calibrated using polypropylene glycol 300 (Applied Biosystems) and had an average mass accuracy of 10–20 ppm. The quantitative analysis of eicosanoids was carried out by LC/MS/MS using a tandem quadrupole mass spectrometer (ABI 4000 Q Trap®, Applied Biosystems), using stable isotope dilution and the multiple-reaction monitoring (MRM) mode. For each sample, a deuterium-labeled internal standard consisting of d4-PGE2 (99 atom % d4; Cayman Chemical, Ann Arbor, MI) was added to the cell supernatant just prior to sample workup. Cellular debris was removed by centrifugation (3,000 g, 10 min). Methanol and acetic acid were added to a final concentration of 10% and 2%, respectively. Eicosanoids were extracted using solid-phase extraction columns (Strata-X; Phenomenex, Rockville, IL). These columns were preconditioned by washing with 2 ml methanol, followed by 2 ml water. The sample was then loaded, and the column washed with 2 ml 0.5% methanol. The eicosanoids were eluted from the column using 1 ml of methanol. This eluate was dried under vacuum and redissolved in 100 μl of solvent system C [water-acetonitrile-formic acid (63:37:0.02; v/v/v)]. Standard curves were prepared in separate experiments in order to relate the abundance of MRM transitions to that of the deuterium-labeled internal standard (10 ng), using primary standards of each eicosanoid (Cayman Chemical). Eicosanoids were separated by reverse-phase HPLC on a reverse-phase C18 column (2.1 mm × 250 mm; Grace-Vydac, Hesperia, CA) operated at a flow rate of 300 μl/min at 25°C. The column was initially equilibrated with 100% solvent C. Samples were then loaded through a 5 μl injection loop and eluted with a linear gradient from 0%–20% solvent D, consisting of acetonitrile-isopropanol (1:1; v/v), in 6 min; next, solvent D was increased to 55% in 6.5 min, and held until 10 min. Finally, solvent system D was linearly increased to 100% in 2 min and held for an additional 1 min. In order to recycle the column, the solvent system was switched to 100% C at 13.5 min and held until 16 min. The mass spectrometer was run in the MRM mode with the electrospray ion source operating in the negative mode (−4.5 kV), with the turbo ion spray source set at 525°C. Collisional activation of each of the eicosanoid precursor ions was carried out using nitrogen as collision gas. Specific MRM pairs are as follows: 11β-PGF2α and PGF2α at m/z 353 → 193 (−80 V); PGE2 and PDG2 at m/z 351 → 189 (−50 V); PGJ2 at m/z 333 → 189 (−30 V); 15D-PGD2 at m/z 333 → 271 (−30 V); 15D-PGJ2 at m/z 315 → 271 (−108 V); and 11-HETE at m/z 319 → 167 (−30 V). The deuterium-labeled internal standards (d4-PGE2 and d4-PGD2) were monitored at m/z 355 → 193. In some cases, the same MRM pairs were used to monitor different eicosanoids (i.e., PGE2 and PGD2); however, because these eluted at different HPLC retention times, they can be analyzed independently. For NMR analysis, a 1 mg sample of Kdo2-Lipid A was dissolved in 0.3 ml CDCl3-CD3OD-D2O (2:3:1; v/v/v) and analyzed by 1H-COSY, TOCSY, and NOESY at 600 MHz, as described previously for lipid A and related compounds (24Ribeiro A.A. Zhou Z. Raetz C.R.H. Multi-dimensional NMR structural analyses of purified lipid X and lipid A (endotoxin).Magn. Reson. Chem. 1999; 37: 620-630Crossref Scopus (50) Google Scholar, 25Que N.L.S. Ribeiro A.A. Raetz C.R.H. Two-dimensional NMR spectroscopy and structures of six lipid A species from Rhizobium etli CE3. Detection of an acyloxyacyl residue in each component and origin of the aminogluconate moiety.J. Biol. Chem. 2000; 275: 28017-28027Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 26Zhou Z. Ribeiro A.A. Raetz C.R.H. High-resolution NMR spectroscopy of lipid A molecules containing 4-amino-4-deoxy-L-arabinose and phosphoethanolamine substituents. Different attachment sites on lipid A molecules from NH4VO3-treated Escherichia coli versus kdsA mutants of Salmonella typhimurium.J. Biol. Chem. 2000; 275: 13542-13551Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). All spectra were recorded at 25°C. RAW 264.7 mouse macrophage tumor cells were plated in DMEM with 10% fetal bovine serum (HyClone Labs; Provo Utah), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). The cells were maintained at 37°C in a humidified 5% CO2 atmosphere. Prior to stimulation, cells were plated at a density of 5 ×105 cells per
To assess the relationship between insulin receptor (IR) kinase activity and insulin action in vivo in humans, we measured glucose disposal rates (GDR) during a series of euglycemic clamp studies. Simultaneously, we measured IR kinase activity in IRs extracted from skeletal muscle obtained by needle biopsy at the end of each clamp. By preserving the phosphorylation state of the receptors as it existed in vivo at the time of biopsy, we could correlate GDR and IR kinase in skeletal muscle. Eight nondiabetic, nonobese male subjects underwent studies at insulin infusion rates of 0, 40, 120, and 1,200 mU/m2 per min. Kinase activity, determined with receptors immobilized on insulin agarose beads, was measured at 0.5 microM ATP, with 1 mg/ml histone, followed by SDS-PAGE. Insulin increased GDR approximately sevenfold with a half-maximal effect at approximately 100 microU/ml insulin and a maximal effect by approximately 400 microU/ml. Insulin also increased IR kinase activity; the half-maximal effect occurred at approximately 500-600 microU/ml insulin with a maximal 10-fold stimulation over basal. Within the physiologic range of insulin concentrations, GDR increased linearly with kinase activation (P less than 0.0006); at supraphysiologic insulin levels, this relationship became curvilinear. Half-maximal and maximal insulin-stimulated GDR occurred at approximately 20 and approximately 50% maximal kinase activation, respectively. These results are consistent with a role of the kinase in insulin action in vivo. Furthermore, they demonstrate the presence of a large amount of "spare kinase" for glucose disposal.
Activation of RAW264.7 cells with a lipopolysaccharide specific for the TLR4 receptor, Kdo 2 ‐Lipid A (KLA), causes a large increase in cellular sphingolipids‐‐from 1.5 to 2.6 × 109 molecules per cell in 24 h, based on the sum of subspecies analyzed by “lipidomic” mass spectrometry. Thus, this study asked: What is the cause of this increase, and is there a cell function connected with it? The sphingolipids arise primarily from de novo biosynthesis; with the exception of ceramide, which is also produced from pre‐existing sources. Nonetheless, the activated RAW264.7 cells have a higher number of sphingolipids per cell because KLA inhibits cell division, thus, the cells are larger and contain increased numbers of membrane vacuoles termed autophagosomes. Indeed, de novo biosynthesis of sphingolipids performs an essential structural and/or signaling function in autophagy because autophagosome formation was eliminated by ISP1 in KLA stimulated RAW264.7 cells; furthermore, an anti‐ceramide antibody co‐localizes with autophagosomes in activated RAW264.7 cells versus the Golgi in unstimulated or ISP1 inhibited cells. These findings establish that KLA induces profound changes in sphingolipid metabolism and content in this macrophage‐like cell line, apparently to produce sphingolipids that are necessary for formation of autophagosomes, which are thought to play important roles in the mechanisms of innate immunity.
