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• W2012885541 abstract "Background: Switching from the acute early initiation to late adaptation response after TLR4 stimulation depends on SirT1.Results: Switching from glucose to fatty acid oxidation between initiation and adaptation responses requires SirT6 and SirT1.Conclusion: Bioenergy integrates metabolism and acute inflammation.Significance: Understanding bioenergy shifts during inflammation may enable development of new therapies. Background: Switching from the acute early initiation to late adaptation response after TLR4 stimulation depends on SirT1. Results: Switching from glucose to fatty acid oxidation between initiation and adaptation responses requires SirT6 and SirT1. Conclusion: Bioenergy integrates metabolism and acute inflammation. Significance: Understanding bioenergy shifts during inflammation may enable development of new therapies. Acute systemic inflammation associated with sepsis involves a sequential shift between the early initiating and later adaptation and immunosuppressive phenotypes (1.Munford R.S. Pugin J. The crucial role of systemic in the innate (non-adaptive) host defense.J. Endotoxin. Res. 2001; 7: 327-332Crossref PubMed Scopus (28) Google Scholar). The early response is brief, severe, and often lethal, but the late phase can persist for weeks with sustained morbidity and mortality from immunosuppression and multiorgan failure (reviewed in Ref. 2.McCall C.E. El Gazzar M. Liu T. Vachharajani V. Yoza B. Epigenetics, bioenergetics, and microRNA coordinate gene-specific reprogramming during acute systemic inflammation.J. Leukoc. Biol. 2011; 90: 439-446Crossref PubMed Scopus (75) Google Scholar). Cellular bioenergy and metabolism play important roles in regulating acute inflammation and immunity. Early innate and adaptive immune responses require a high energy state supported by glucose-dependent production of ATP and activation of NADPH oxidase to kill microorganisms by reactive oxygen species (3.Schumer W. Metabolic aspects of shock.Surg. Annu. 1974; 6: 1-16PubMed Google Scholar). During this early stage of sepsis, precipitous decreases in ATP production by mitochondrial oxidative phosphorylation occur (4.Bolaños J.P. Almeida A. Moncada S. Glycolysis. A bioenergetic or a survival pathway?.Trends Biochem. Sci. 2010; 35: 145-149Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), and glycolysis provides the primary source of ATP (4.Bolaños J.P. Almeida A. Moncada S. Glycolysis. A bioenergetic or a survival pathway?.Trends Biochem. Sci. 2010; 35: 145-149Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 5.Carré J.E. Orban J.C. Re L. Felsmann K. Iffert W. Bauer M. Suliman H.B. Piantadosi C.A. Mayhew T.M. Breen P. Stotz M. Singer M. Survival in Critical Illness is Associated with Early Activation of Mitochondrial Biogenesis.Am. J. Respir. Crit. Care Med. 2010; 182: 745-751Crossref PubMed Scopus (271) Google Scholar). In contrast, the later stage of acute inflammation is a low-energy response (6.Singer M. Cellular dysfunction in sepsis.Clin. Chest Med. 2008; 29: 655-660Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) that heals and eventually restores homeostasis. However, it also represses innate and adaptive immunity (7.Hotchkiss R.S. Coopersmith C.M. McDunn J.E. Ferguson T.A. The sepsis seesaw. Tilting toward immunosuppression.Nat. Med. 2009; 15: 496-497Crossref PubMed Scopus (436) Google Scholar), as recently documented in human spleen and lung from deceased sepsis patients (8.Boomer J.S. To K. Chang K.C. Takasu O. Osborne D.F. Walton A.H. Bricker T.L. Jarman 2nd, S.D. Kreisel D. Krupnick A.S. Srivastava A. Swanson P.E. Green J.M. Hotchkiss R.S. Immunosuppression in patients who die of sepsis and multiple organ failure.JAMA. 2011; 306: 2594-2605Crossref PubMed Scopus (1125) Google Scholar). A caution from this important study is that postmortem changes in the viable cells studied might change phenotype. Emerging data support that interactions between inflammation and metabolism play critical roles in chronic inflammatory diseases like obesity with diabetes and atherosclerosis (9.Chawla A. Nguyen K.D. Goh Y.P. Macrophage-mediated inflammation in metabolic disease.Nat. Rev. Immunol. 2011; 11: 738-749Crossref PubMed Scopus (958) Google Scholar, 10.Odegaard J.I. Ricardo-Gonzalez R.R. Goforth M.H. Morel C.R. Subramanian V. Mukundan L. Red Eagle A. Vats D. Brombacher F. Ferrante A.W. Chawla A. Macrophage-specific PPARγ controls alternative activation and improves insulin resistance.Nature. 2007; 447: 1116-1120Crossref PubMed Scopus (1588) Google Scholar, 11.Olefsky J.M. Glass C.K. Macrophages, inflammation, and insulin resistance.Annu. Rev. Physiol. 2010; 72: 219-246Crossref PubMed Scopus (1977) Google Scholar). These chronic inflammatory and metabolic responses are heterogeneous and involve cells of both innate and adaptive immunity. Less is known about how metabolism influences acute inflammation, which progresses through sequential stages. We recently reported that NAD+ informs deacetylase sirtuin 1 (SirT1) 3The abbreviations used are: SirT1deacetylase sirtuin 1TLRToll-like receptorCLPcecal ligation and puncturePDHKpyruvate dehydrogenase kinasePDHApyruvate dehydrogenase EA1PFKphospho-fructose kinaseCPTcarnitine palmitoyl transferasePGCperoxisome proliferator-activated receptor γ coactivatorNamptnicotinamide phosphoribosyltransferaseHIFhypoxia-inducible factor. to direct a sequential epigenetic switch between early and late TLR4 responses in a THP-1 promonocyte sepsis cell model and in human sepsis blood leukocytes (12.Liu T.F. Yoza B.K. El Gazzar M. Vachharajani V.T. McCall C.E. NAD+-dependent SIRT1 deacetylase participates in epigenetic reprogramming during endotoxin tolerance.J. Biol. Chem. 2011; 286: 9856-9864Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). In this study, we used the same THP-1 sepsis model of TLR4 responses, as well as human and murine sepsis leukocytes to show that NAD+-dependent sensing by SirT1 and SirT6 integrates sequential reprogramming of metabolic and acute inflammatory responses. deacetylase sirtuin 1 Toll-like receptor cecal ligation and puncture pyruvate dehydrogenase kinase pyruvate dehydrogenase EA1 phospho-fructose kinase carnitine palmitoyl transferase peroxisome proliferator-activated receptor γ coactivator nicotinamide phosphoribosyltransferase hypoxia-inducible factor. Blood was drawn from healthy controls and sepsis subjects according to the Institutional Review Board protocol approved by Wake Forest University School of Medicine. Patients fit the diagnosis of the systemic inflammatory response syndrome with septic shock and were receiving vasopressors (13.Levy M.M. Fink M.P. Marshall J.C. Abraham E. Angus D. Cook D. Cohen J. Opal S.M. Vincent J.L. Ramsay G. 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference.Crit. Care Med. 2003; 31: 1250-1256Crossref PubMed Scopus (4655) Google Scholar). Blood leukocytes were isolated as described (12.Liu T.F. Yoza B.K. El Gazzar M. Vachharajani V.T. McCall C.E. NAD+-dependent SIRT1 deacetylase participates in epigenetic reprogramming during endotoxin tolerance.J. Biol. Chem. 2011; 286: 9856-9864Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The blood leukocytes were 95% viable and subjected to cell culture under the indicated conditions. We used mixed blood leukocytes because both neutrophils and mononuclear cells form silent heterochromatin during the late stage of sepsis that exhibits immunosuppression. The sublethal animal sepsis model was approved by the Institutional Animal Care and Use Committee of the Wake Forest University School of Medicine, and studies were performed according to the National Institutes of Health guidelines. C57BL/6, 6- to 8-week-old mice were obtained from The Jackson Laboratories (Bar Harbor, ME), and the cecal ligation and puncture (CLP) procedure was performed under anesthetization as detailed previously (14.Vachharajani V. Cunningham C. Yoza B. Carson Jr., J. Vachharajani T.J. McCall C. Adiponectin Deficiency exaggerates sepsis-induced microvascular dysfunction in the mouse brain.Obesity. 2012; 20: 498-504Crossref PubMed Scopus (35) Google Scholar). In sham mice, all procedures were identical to the CLP mice except for the cecal ligation and puncture. The LD50 of septic mice is 77 h after CLP. Splenocytes from septic mice contain a mixture myeloid-derived suppressor cells that are hyporesponsive to LPS stimulation ex vivo by 18 h after CLP (unpublished observations) 4T. F. Liu, V. T. Vachharajani, B. K. Yoza, and C. E. McCall, unpublished observations.. Thus, the spleen cell phenotype in mice mimics the circulating mixed leukocyte phenotype observed in human sepsis. However, there are two potential limitations from using mixtures of blood or spleen cells obtained from normal or sepsis participants. One is the presence of mixed cell types (neutrophils, monocyte/macrophages, and T or B lymphocytes). The second is that the state of cell differentiation (e.g. immaturity) may differ. A possible advantage of impure populations is the cross-talk that may occur during inflammation (e.g. paracrine effects). THP-1 cells were obtained from the ATCC and maintained in RPMI 1640 medium (Invitrogen) supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, and 10% fetal bovine serum (HyClone, Logan, UT) in a humidified incubator with 5% CO2 at 37 °C. Cells were stimulated with 1 μg/ml of Gram-negative bacteria LPS for indicated times to generate different inflammatory phases. This ultrapure LPS product extracted from Escherichia coli serotype 0111:B4 (Sigma) only acts via TLR4 (15.Imler J.L. Hoffmann J.A. Toll signaling. The TIReless quest for specificity.Nat. Immunol. 2003; 4: 105-106Crossref PubMed Scopus (63) Google Scholar, 16.Yumoto H. Chou H.H. Takahashi Y. Davey M. Gibson 3rd, F.C. Genco C.A. Sensitization of human aortic endothelial cells to lipopolysaccharide via regulation of Toll-like receptor 4 by bacterial fimbria-dependent invasion.Infect. Immun. 2005; 73: 8050-8059Crossref PubMed Scopus (58) Google Scholar). We have confirmed TLR4-dependent responsivity of LPS in murine macrophages not expressing TLR4 or TLR2 (unpublished observations 5E. Lorenz and C. E. McCall, unpublished observations.). In this model of sepsis responses, the early inflammatory response is assessed at 4–8 h after TLR4 stimulation. The later adaptation stage is present by 24–48 h after TLR4 stimulation and mimics LPS responsivity of sepsis blood leukocytes. In some experiments, cells were pretreated 24 h with 10 nm FK866 (Cayman Chemical) (to deplete cellular NAD+), 10 mm 2-deoxyglucose (2-DG), 1 μm echinomycin (HIF-1α inhibitor), or 10 nm Etomoxir (carnitine palmitoyl transferase 1 inhibitor). The same quantity of viable cells as determined by trypan blue exclusion is used for each following experimental treatment after LPS, inhibitors, or electronic transfection. Uptake of glucose and fatty acid were measured by radiolabel (17.Wu X. Motoshima H. Mahadev K. Stalker T.J. Scalia R. Goldstein B.J. Involvement of AMP-activated protein kinase in glucose uptake stimulated by the globular domain of adiponectin in primary rat adipocytes.Diabetes. 2003; 52: 1355-1363Crossref PubMed Scopus (389) Google Scholar, 18.Gao J. Ye H. Serrero G. Stimulation of adipose differentiation-related protein (ADRP) expression in adipocyte precursors by long-chain fatty acids.J. Cell Physiol. 2000; 182: 297-302Crossref PubMed Scopus (97) Google Scholar). One million cells in 100 μl were starved in triplicate in polypropylene vials for 30 min at 37 °C in glucose-free or serum-free Hanks' buffer. The assay was initiated by the addition of another 100 μl of hot buffer containing 1 μCi of D-[6-14C]glucose (PerkinElmer LifeSciences) and 2.5 μm cold glucose or 1 μCi of 1-[14C]palmitic acid in 0.2% BSA-Hanks' buffer. Glucose transport reaction was terminated after 5 min by washing cells three times in ice-cold PBS containing cytochalasin B (Sigma). Fatty acid uptake was stopped by washing cells with ice-cold PBS containing 0.1% BSA and 200 mm phloretin (Sigma). Cell pellets were solubilized in 0.5 m NaOH, and extracts were neutralized by glacial acetic acid. Cell-associated radioactivity was determined by scintillation β counter. Central wells containing 1 million nutrients-starved cells in triplicates were placed into scintillation tube. After addition of 1 μCi of D-[6-14C]glucose and 2.5 μm cold glucose or 1 μCi of 1-[14C]palmitic acid in 0.2% BSA-Hank's buffer to cells, the scintillation tubes were sealed by a rubber stopper. Cells were incubated at 37 °C in a water bath with rotation. After 1 h of incubation, 200 μl of 2 N HCl was injected into the central well to terminate metabolic reactions, and 500 μl of Hyamine (PerkinElmer Life Sciences) was injected into the scintillation tube. After overnight shaking at room temperature, the central well was removed and 14CO2 generated by the oxidation of D-[6-14C]glucose or 1-[14C]palmitic acid was detected using β counter. One μCi of D-[6-14C]glucose alone or 1-[14C]palmitic acid alone in same amount of buffer was set for background counts. Glycolysis was measured by the conversion of D-[5-3H(N)]glucose to tritiated water (19.Sambandam N. Lopaschuk G.D. AMP-activated protein kinase (AMPK) control of fatty acid and glucose metabolism in the ischemic heart.Prog. Lipid Res. 2003; 42: 238-256Crossref PubMed Scopus (142) Google Scholar). Cells in central wells in glucose-free RPMI (Invitrogen) in triplicates were incubated with 1 μCi of D-[5-3H(N)]glucose (PerkinElmer Life Sciences) at 37 °C for 1 h in scintillation tubes containing 1 ml of H2O. The reaction was stopped by adding HCl (1 N final), and the scintillation tube was sealed. [3H]2O generated by enolase activity from D-[5-3H(N)]glucose was vaporized overnight in a 50 °C oven and cooled down overnight at 4 °C. After removal of the central wells, [3H]2O was counted for detection of the glycolytic rate. 1 μCi of D-[5-3H(N)]glucose alone in triplicates was set for background control. 1 μCi of [3H]2O (PerkinElmer Life Sciences) alone in triplicates was set for detection the efficiency of this water vapor exchange. Levels of human TNF-α, IL-10, and RelB mRNA were measured by quantitative real-time RT-PCR using gene-specific TaqMan primer/probe sets in an ABI prism 7000 sequence detection system (Applied Biosystems). GAPDH mRNA was the internal loading control. For knockdown, 60 pmol of a pool of three target-specific siRNA (Santa Cruz Biotechnology) were electronically transfected into responsive THP-1 cells for 24 h using Amaxa nucleofector kit V and an Amaxa nucleofector II device (Lonza, Inc.) and with 1 μg/ml LPS before harvest. A pool of scrambled siRNAs was transfected as a negative control. Equal amounts (50 μg) of cell lysates were separated by SDS-PAGE electrophoresis and transferred to a polyvinylidene difluoride membrane (PerkinElmer Life Sciences). Blots were blocked with 5% milk-TBS-Tween 20 for 1 h at room temperature and probed overnight at 4 °C with primary antibodies against Glut1 (Abcam), pyruvate dehydrogenase kinase 1 (PDHK1) (Enzo Life Sciences), pyruvate dehydrogenase E1α1 (PDHA1) (Invitrogen), p-PDHA1-Ser-232 (Calbiochem), phospho-fructose kinase 1 (pFK1) and pFK2 (Novus Biologicals), lactate dehydrogenase (Fitzgerald), CD36 (Thermo Scientific), carnitine palmitoyl transferase 1 (CPT-1), peroxisome proliferator-activated receptor γ coactivator 1-α and β (PGC-1α and PGC-1β) (Santa Cruz Biotechnology), and SirT1 and SirT6 (Cell Signaling Technology). β-actin was used as a loading control and was probed with mouse anti-human β-actin monoclonal antibody (Sigma). Protein complexes were detected by incubation for 1 h at room temperature with secondary antibody conjugated to horseradish peroxidase (Sigma) diluted at 1:5000 in blocking buffer and then detected by Enhanced Chemiluminescence Plus (GE Healthcare). Aliquots of 1 million cells were pelleted at 600 × g and resuspended in 80 μl of PBS with 1% BSA. Different cell aliquots were incubated with 10 μl of carboxyfluorescein-conjugated mouse IgG1 anti-human Glut1 antibody (Sigma), FITC-conjugated mouse IgG1anti-human CD36 antibody or isotype control antibody (Stem Cell Technologies), respectively. After incubation for 1 h on ice, cells were washed with PBS/BSA and resuspended in 1 ml of PBS/BSA. A volume of 250 μl of 3.7% formaldehyde was added to each tube, and the cells were assayed on an EPICS-XL flow cytometer (Coulter, Hialeah, FL) with filters set for FITC fluorescence detection. Gates were based on the basis of isotype control antibody staining so that there were <2% positive cells with isotype control antibodies. Ten thousand cells were evaluated for FITC positivity. Data were analyzed using FlowJo software. For Glut1 staining, cells were incubated with 1 μg/ml of mouse anti-human Glut1 monoclonal antibody (R&D Systems) for 1 h, washed, and incubated for another 1 h on ice with rhodamine-conjugated goat anti-mouse IgG1 (1–1000 diluted in blocking buffer, Sigma). CD36 were stained with FITC-anti-human CD36 monoclonal antibody (1:10 diluted in blocking buffer, Stemcell Technologies) for 1 h. After two more washes with PBS, 1000 cells were fixed with 3.7% formaldehyde-PBS, cytospun onto slides, mounted (Victor Labs, Burlingame, CA), and analyzed using an LSM 510 microscope (Zeiss) with the LSM 510 image browser software. Intracellular glycogen was stained with the periodic acid Schiff procedure. Cells were cytospinned onto slides and fixed with Carnoy's fixative buffer containing 60% alcohol, 30% chloroform, and 10% glacial acetic acid. After washing with deionized water, slides were stained with 0.5% (w/v) of periodic acid solution (Sigma) for 10 min followed by Schiff reagent (Sigma) for 5 min. Slides were then counterstained with hematoxylin and examined using a Zeiss Axiocam charge-coupled device camera. Total cellular NADt (NAD+ + NADH) extraction and evaluation were performed using a colorimetric NAD+/NADH assay kit (BioVision) according to the instructions of the manufacturer. NADt extractions were filtered by passing samples through a 10 Kd molecular weight cutoff filter to exclude the possible NADH consuming enzymes. For cellular NADH detection, NAD+ was decomposed before reaction by heating NADt samples for 30 min at 60 °C. Fifty microliters of NADt, NADH samples, or NADH standard were mixed for 10 min with 100 μl of working reagent in duplicate in a 96-well plate. 10 μl of NADH developer reagent was added to each well and incubated for another 1 h. The optical density was read at 450 nm. Cellular NADt and NADH levels of unknown samples were calculated from the standard curve and analyzed by Prism software (GraphPad Prism, version 4.0, GraphPad Software, San Diego, CA) and were normalized against protein levels. NAD+ levels were obtained by subtraction of the NADH level from NADt. The ratio of NAD+/NADH was calculated as (NADt-NADH)/NADH. Human and mouse plasma were prepared by centrifugation of blood samples to remove cell components. Plasma pyruvate levels were measured using EnzyChromTM pyruvate assay kit (Bioassy Systems) according to the instructions of the manufacturer. Briefly, plasma was diluted 2-fold in PBS. 10 μl of samples or pyruvate standards were transferred into 96-well plates in duplicates. 90 μl of working solution was added into each well and incubated for 30 min at room temperature. The optical density at 570 nm was read, and the concentration of plasma pyruvate was calculated from a standard curve. Differences of metabolic changes between two related conditions were analyzed by Student unpaired “t” test, and the temporal changes in NAD+, NADH and NAD+/NADH ration following TLR stimulation were analyzed by ANOVA using GraphPad Prism version 4 (San Diego, CA). p values of less than 0.05 were considered significant. We have, over 20 years, found that TLR4-activated THP-1 human monocytic cells simulate sequential innate inflammatory responses of human sepsis blood leukocytes on the basis of gene-specific formation of silenced heterochromatin at acute proinflammatory genes and the presence of RelB-specific generation of adaptation and LPS tolerance during late acute inflammatory responses (20.LaRue K.E. McCall C.E. A labile transcriptional repressor modulates endotoxin tolerance.J. Exp. Med. 1994; 180: 2269-2275Crossref PubMed Scopus (99) Google Scholar). Here, we further substantiate the temporal sequence of accentuated to repressed expression of proinflammatory TNF-α to immunorepressive IL-10 following TLR4 stimulation of THP-1 cells (Fig. 1A). To determine whether cellular metabolism varies with sequential TLR4 responses, we examined glucose and fatty acid metabolism. We used D-[6-14C]glucose to follow glucose uptake and glucose oxidation, D-[5-3H (N)]glucose for glycolysis analysis, and 1-[14C]palmitic acid for analysis of fatty acid uptake and oxidation. To validate the radiolabeling metabolic assays, we found that 500-fold excess of cold D-glucose or palmitate blocks uptakes of D-[6-14C]glucose and 1-[14C]palmitate up to 96% and 92%, respectively (not shown). We found that glucose uptake increased for 8 h (early acute inflammation phase) and decreased by 24 h (adaptation phase) (Fig. 1B). In contrast, glucose oxidation deceased rapidly and remained low at 24 h (Fig. 1C). We confirmed that increased glucose flux and decreased glucose oxidation during early TLR4 responses reflected enhanced glycolysis (Fig. 1D). Next, we showed that fatty acid uptake was relatively unchanged during the early TLR4 response but significantly increased by 24 h (Fig. 1E). This increase paralleled elevated fatty acid oxidation during the adaptation stage (Fig. 1F). Thus, increased glycolysis dominates early TLR4 responses and fatty acid flux and oxidation dominate late responses in THP-1 cells. Distinct signaling events control glucose and fatty acid metabolism (21.Vats D. Mukundan L. Odegaard J.I. Zhang L. Smith K.L. Morel C.R. Wagner R.A. Greaves D.R. Murray P.J. Chawla A. Oxidative metabolism and PGC-1β attenuate macrophage-mediated inflammation.Cell Metab. 2006; 4: 13-24Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar) (Fig. 2A). We further observed that the Warburg-like glycolytic profile during early acute TLR4 responses is supported by 1) the increased expression of glucose transporter Glut1 and its cell surface translocation, 2) deactivation of mitochondrial PDHA1, and 3) increased expression of glycolytic enzymes pFK1 and pFK2 and accumulation of cellular pyruvate and lactate (Figs. 2, B–G). We also observed that glycogenolysis supports glycolysis during late inflammatory response when Glut1 expression is diminished (data not shown). Increased fatty acid oxidation was associated with increased expression of CD36, a member of the class B scavenger receptor/transporter with a high affinity for long chain fatty acids, and CPT-1, the rate-limiting enzyme for long chain fatty acid mitochondrial transporter and fatty acid oxidation (Fig. 2, H–J). The TLR4-induced expression of CD36 and CPT-1 shown by Western blot analysis was further confirmed by densitometry analysis (Fig. 2H, right panel). We reported previously that Nampt-dependent accumulation of cellular NAD+ coordinates epigenetic reprogramming when TLR4 responses switch from early to late phenotypes (Fig. 3A) (12.Liu T.F. Yoza B.K. El Gazzar M. Vachharajani V.T. McCall C.E. NAD+-dependent SIRT1 deacetylase participates in epigenetic reprogramming during endotoxin tolerance.J. Biol. Chem. 2011; 286: 9856-9864Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Following early TLR4 activation, cellular NAD+ levels were decreased and then steadily accumulated. Mirroring the high energy requirement, TLR4 also increased NADH levels that later decreased (Fig. 3B). The dynamic changes in cellular NAD+ and NADH levels following TLR response are statistically significant (p = 0.0016 and p = 0.0002, respectively). NAD+/NADH ratios increased (p = 0.0004, Fig. 3C). We then tested whether NAD+-dependent events also regulate the metabolic switch during the sequential TLR4 inflammatory response. To do this, we depleted NAD+ by overnight pretreatment of cells with Nampt-specific inhibitor FK866 followed by TLR4 stimulation for 24 h. FK866 pretreatment decreased the basal rate of glucose oxidation and could not further diminish it without NAD+ after TLR4 activation (Fig. 3D). The reduction of basal glucose oxidation after FK866 treatment could have resulted from the decreased PDHA1 expression. However, inhibition of Nampt activity almost totally blocked late-state TLR4-induced increases in fatty acid oxidation, although the basal rate of fatty acid β oxidation was not altered (Fig. 3E). These observations parallel the early to late epigenetic switching response (12.Liu T.F. Yoza B.K. El Gazzar M. Vachharajani V.T. McCall C.E. NAD+-dependent SIRT1 deacetylase participates in epigenetic reprogramming during endotoxin tolerance.J. Biol. Chem. 2011; 286: 9856-9864Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The NAD+-dependent metabolic switch is further supported by the increased expression of PDHK1 and the phosphorylation of PDHA1 at PDHK1-specific site PDHA1-Ser-232, the inactive form of PDHA1, and decreased expression of PDHA1 and CPT-1 proteins in the presence of FK866 (Fig. 3F). We then reasoned that NAD+ sensors SirT6 and SirT1 might differentially coordinate the metabolic switching between early and late acute inflammatory responses because SirT6 regulates glycolysis and SirT1 regulates fatty acid oxidation (22.Zhong L. Mostoslavsky R. SIRT6. A master epigenetic gatekeeper of glucose metabolism.Transcription. 2010; 1: 17-21Crossref PubMed Scopus (73) Google Scholar). We first examined levels of SirT1 and SirT6 protein and confirmed our previous observation that SirT1 increases after TLR4 stimulation (12.Liu T.F. Yoza B.K. El Gazzar M. Vachharajani V.T. McCall C.E. NAD+-dependent SIRT1 deacetylase participates in epigenetic reprogramming during endotoxin tolerance.J. Biol. Chem. 2011; 286: 9856-9864Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). In contrast, SirT6 was constitutively expressed, decreased after early TLR4 stimulation, and moderately increased during the late adaptation phase (Fig. 4A). We then assessed effects of SirT1 or SirT6 depletion on glucose and fatty acid metabolism. To do this, THP-1 cells were transfected with either control or test siRNAs for 24 h and analyzed 24 h after TLR4 stimulation. Immunoblot analysis showed that SirT6 knockdown caused 90% reduction of SirT6 protein without changing SirT1 levels. In contrast, SirT1 knockdown depleted both itself and SirT6, confirming reports that SirT1 regulates SirT6 (23.Kim H.S. Xiao C. Wang R.H. Lahusen T. Xu X. Vassilopoulos A. Vazquez-Ortiz G. Jeong W.I. Park O. Ki S.H. Gao B. Deng C.X. Hepatic-specific disruption of SIRT6 in mice results in fatty liver formation due to enhanced glycolysis and triglyceride synthesis.Cell Metab. 2010; 12: 224-236Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar) (Fig. 4H). SirT6 depletion significantly increased glucose uptake and glycolysis and further decreased glucose oxidation (Fig. 4, B–D) but did not alter fatty acid uptake or oxidation (Fig. 4, E and F). In contrast, SirT1 knockdown significantly decreased fatty acid uptake and fatty acid oxidation without changing glucose uptake, glycolysis, or glucose oxidation after TLR4 stimulation. Thus, both glucose and fatty acid metabolic changes during the acute TLR4 inflammatory responses depend on NAD+ for sirtuin activation, but SirT6 predominantly opposes the glucose metabolic switch, and SirT1 primarily supports the fatty acid switch. The differentially regulated inflammatory metabolic switch by SirT1 and SirT6 was further supported by immunoflourescence examination of cell surface transporters Glut1 and CD36. Glut1 transporter levels increased in early TLR4 responses and diminished by the 24 h late stage. In contrast, CD36 expression significantly increased by 24 h (Fig. 4G, upper panel). SirT1 knockdown partially blocked the decrease in Glut1 expression but completely attenuated increased CD36 expression (Fig. 4G, center panel). In contrast, SirT6 knockdown limited Glut1 decreases observed at 24 h without affecting CD36 (Fig. 4G, bottom panel). Immunoblot analysis of Glut1 and CD36 expression confirmed the immunoflourescence observations after gene-specific knockdowns (Fig. 4H). HIF-1α is a master regulator of genes controlling glycolysis (24.Weidemann A. Johnson R.S. Biology of HIF-1α.Cell Death. Differ. 2008; 15: 621-627Crossref PubMed Scopus (624) Google Scholar) and PGC-1 of genes controlling fatty acid oxidation and mitochondrial biogenesis (25.Gleyzer N. Scarpulla R.C. PGC-1-related coactivator (PRC), a sensor of metabolic stress, orchestrates a redox-sensitive program of inflammatory gene expression.J. Biol. Chem. 2011; 286: 39715-39725Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). SirT6 represses the function of HIF-1α by epigenetically silencing chromatin at HIF-1α target gene promoters, thereby balancing glucose metabolism (26.Zhong L. D'Urso A. Toiber D. Sebastian C. Henry R.E. Vadysirisack D.D. Guimaraes A. Marinelli B. Wikstrom J.D. Nir T. Clish C.B. Vaitheesvaran B. Iliopoulos O. Kurland I. Dor Y. Weissleder R. Shirihai O.S. Ellisen L.W. Espinosa J.M. Mostoslavsky R. The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1α.Cell. 2010; 140: 280-293Abstract Full Text Full Text PDF PubMed Scopus (787) Google Scholar). In contrast, SirT1 directly binds and activates PGC-1 coactivators to support the fatty acid oxidative pathway and mitochondrial biogenesis (27.Rodgers J.T. Lerin C. Haas W. Gygi S.P. Spie" @default.
• W2012885541 created "2016-06-24" @default.
• W2012885541 creator A5024933730 @default.
• W2012885541 creator A5038338463 @default.
• W2012885541 creator A5040430463 @default.
• W2012885541 creator A5047444154 @default.
• W2012885541 date "2012-07-01" @default.
• W2012885541 modified "2023-10-16" @default.
• W2012885541 title "NAD+-dependent Sirtuin 1 and 6 Proteins Coordinate a Switch from Glucose to Fatty Acid Oxidation during the Acute Inflammatory Response" @default.
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