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Adipocyte mTORC1 deficiency promotes adipose tissue inflammation and NLRP3 inflammasome activation via oxidative stress and de novo ceramide synthesis
2017; Elsevier BV; Volume: 58; Issue: 9 Linguagem: Inglês
10.1194/jlr.m074518
ISSN1539-7262
AutoresPatrícia Chimin, Maynara L. Andrade, Thiago Belchior, Vivian A. Paschoal, Juliana Magdalon, Alex Shimura Yamashita, Érique Castro, Ângela Castoldi, Adriano B. Chaves‐Filho, Marcos Y. Yoshinaga, Sayuri Miyamoto, Niels Olsen Saraiva Câmara, William T. Festuccia,
Tópico(s)Eicosanoids and Hypertension Pharmacology
ResumoMechanistic target of rapamycin complex (mTORC)1 activity is increased in adipose tissue of obese insulin-resistant mice, but its role in the regulation of tissue inflammation is unknown. Herein, we investigated the effects of adipocyte mTORC1 deficiency on adipose tissue inflammation and glucose homeostasis. For this, mice with adipocyte raptor deletion and controls fed a chow or a high-fat diet were evaluated for body mass, adiposity, glucose homeostasis, and adipose tissue inflammation. Despite reducing adiposity, adipocyte mTORC1 deficiency promoted hepatic steatosis, insulin resistance, and adipose tissue inflammation (increased infiltration of macrophages, neutrophils, and B lymphocytes; crown-like structure density; TNF-α, interleukin (IL)-6, and monocyte chemoattractant protein 1 expression; IL-1β protein content; lipid peroxidation; and de novo ceramide synthesis). The anti-oxidant, N-acetylcysteine, partially attenuated, whereas treatment with de novo ceramide synthesis inhibitor, myriocin, completely blocked adipose tissue inflammation and nucleotide oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3)-inflammasome activation, but not hepatic steatosis and insulin resistance induced by adipocyte raptor deletion. Rosiglitazone treatment, however, completely abrogated insulin resistance induced by adipocyte raptor deletion. In conclusion, adipocyte mTORC1 deficiency induces adipose tissue inflammation and NLRP3-inflammasome activation by promoting oxidative stress and de novo ceramide synthesis. Such adipose tissue inflammation, however, is not an underlying cause of the insulin resistance displayed by these mice. Mechanistic target of rapamycin complex (mTORC)1 activity is increased in adipose tissue of obese insulin-resistant mice, but its role in the regulation of tissue inflammation is unknown. Herein, we investigated the effects of adipocyte mTORC1 deficiency on adipose tissue inflammation and glucose homeostasis. For this, mice with adipocyte raptor deletion and controls fed a chow or a high-fat diet were evaluated for body mass, adiposity, glucose homeostasis, and adipose tissue inflammation. Despite reducing adiposity, adipocyte mTORC1 deficiency promoted hepatic steatosis, insulin resistance, and adipose tissue inflammation (increased infiltration of macrophages, neutrophils, and B lymphocytes; crown-like structure density; TNF-α, interleukin (IL)-6, and monocyte chemoattractant protein 1 expression; IL-1β protein content; lipid peroxidation; and de novo ceramide synthesis). The anti-oxidant, N-acetylcysteine, partially attenuated, whereas treatment with de novo ceramide synthesis inhibitor, myriocin, completely blocked adipose tissue inflammation and nucleotide oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3)-inflammasome activation, but not hepatic steatosis and insulin resistance induced by adipocyte raptor deletion. Rosiglitazone treatment, however, completely abrogated insulin resistance induced by adipocyte raptor deletion. In conclusion, adipocyte mTORC1 deficiency induces adipose tissue inflammation and NLRP3-inflammasome activation by promoting oxidative stress and de novo ceramide synthesis. Such adipose tissue inflammation, however, is not an underlying cause of the insulin resistance displayed by these mice. Chronic low-intensity inflammation is an important linking factor between visceral obesity and associated metabolic complications such as insulin resistance (1.Hotamisligil G.S. 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Viguerie N. et al.Adipose tissue transcriptomic signature highlights the pathological relevance of extracellular matrix in human obesity.Genome Biol. 2008; 9: R14Crossref PubMed Scopus (323) Google Scholar, 4.Hotamisligil G.S. Erbay E. Nutrient sensing and inflammation in metabolic diseases.Nat. Rev. Immunol. 2008; 8: 923-934Crossref PubMed Scopus (782) Google Scholar). One important proinflammatory event triggered by TLR4-IKK-NFκB signaling upon obesity is the activation of nucleotide oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3)-inflammasome, a multimeric cytosolic protein complex activated by pathogen- and danger-associated molecular patterns, that promotes caspase 1 (CASP1)-mediated interleukin (IL)-1β processing and secretion (5.Vandanmagsar B. Youm Y.H. Ravussin A. Galgani J.E. Stadler K. Mynatt R.L. Ravussin E. Stephens J.M. Dixit V.D. The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance.Nat. 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Mitochondrial reactive oxygen species (ROS), saturated fatty acids, and cholesterol crystals, among others, were shown to induce inflammasome priming, whereas ceramides, potassium efflux, and mitochondrial DNA induce inflammasome assembly and activation (6.Henao-Mejia J. Elinav E. Thaiss C.A. Flavell R.A. Inflammasomes and metabolic disease.Annu. Rev. Physiol. 2014; 76: 57-78Crossref PubMed Scopus (96) Google Scholar). Obesity, through unknown mechanisms, is also associated with a chronic activation of mechanistic target of rapamycin complex (mTORC)1 in adipose tissue (7.Um S.H. Frigerio F. Watanabe M. Picard F. Joaquin M. Sticker M. Fumagalli S. Allegrini P.R. Kozma S.C. Auwerx J. et al.Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity.Nature. 2004; 431: 200-205Crossref PubMed Scopus (1363) Google Scholar). mTORC1, which is composed of mammalian lethal with Sec13 protein 8 (mLST8), Tti1/Tel2 complex, DEP domain-containing mTOR-interacting protein (DEPTOR), regulatory-associated protein of mTOR (raptor), proline-rich Akt substrate 40 kDa (PRAS40), and the serine/threonine kinase mTOR as its catalytic center, is mainly activated by amino acids and growth factors and regulates protein and lipid syntheses, autophagy, and adipose tissue mass and metabolism (8.Laplante M. Sabatini D.M. mTOR signaling in growth control and disease.Cell. 2012; 149: 274-293Abstract Full Text Full Text PDF PubMed Scopus (6164) Google Scholar). Specifically regarding the regulation of adiposity, recent findings obtained from mice with adipocyte mTORC1 constitutive activation [tuberous sclerosis 1 (Tsc1) deletion] (9.Magdalon J. Chimin P. Belchior T. Neves R.X. Vieira-Lara M.A. Andrade M.L. Farias T.S. Bolsoni-Lopes A. Paschoal V.A. Yamashita A.S. et al.Constitutive adipocyte mTORC1 activation enhances mitochondrial activity and reduces visceral adiposity in mice.Biochim. Biophys. Acta. 2016; 1861: 430-438Crossref PubMed Scopus (30) Google Scholar) or deficiency (raptor deletion) (10.Polak P. Cybulski N. Feige J.N. Auwerx J. Rüegg M.A. Hall M.N. Adipose-specific knockout of raptor results in lean mice with enhanced mitochondrial respiration.Cell Metab. 2008; 8: 399-410Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 11.Lee P.L. Tang Y. Li H. Guertin D.A. Raptor/mTORC1 loss in adipocytes causes progressive lipodystrophy and fatty liver disease.Mol. Metab. 2016; 5: 422-432Crossref PubMed Scopus (69) Google Scholar) or mild inhibition (DEPTOR overexpression) (12.Laplante M. Horvat S. Festuccia W.T. Birsoy K. Prevorsek Z. Efeyan A. Sabatini D.M. DEPTOR cell-autonomously promotes adipogenesis, and its expression is associated with obesity.Cell Metab. 2012; 16: 202-212Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) indicate that optimal levels of mTORC1 activity, neither too high nor too low, are required for its pro-fat accretion actions (9.Magdalon J. Chimin P. Belchior T. Neves R.X. Vieira-Lara M.A. Andrade M.L. Farias T.S. Bolsoni-Lopes A. Paschoal V.A. Yamashita A.S. et al.Constitutive adipocyte mTORC1 activation enhances mitochondrial activity and reduces visceral adiposity in mice.Biochim. Biophys. Acta. 2016; 1861: 430-438Crossref PubMed Scopus (30) Google Scholar). Interestingly, in spite of markedly reducing adiposity, adipocyte raptor deletion (mTORC1 deficiency) using either ap2- or adiponectin-cre has resulted in different glucose homeostasis outcomes. Indeed, while ap2-cre-driven raptor deletion protected mice from diet-induced obesity, glucose intolerance, and insulin resistance, promoted adipose tissue browning, and increased energy expenditure (10.Polak P. Cybulski N. Feige J.N. Auwerx J. Rüegg M.A. Hall M.N. Adipose-specific knockout of raptor results in lean mice with enhanced mitochondrial respiration.Cell Metab. 2008; 8: 399-410Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar), adiponectin-cre-driven raptor deletion induced hepatic steatosis and insulin resistance, as evidenced by fasting hyperinsulinemia and impaired insulin tolerance (11.Lee P.L. Tang Y. Li H. Guertin D.A. Raptor/mTORC1 loss in adipocytes causes progressive lipodystrophy and fatty liver disease.Mol. Metab. 2016; 5: 422-432Crossref PubMed Scopus (69) Google Scholar). Changes in adiposity are generally associated with alterations in adipose tissue-resident leukocyte content and profile. Indeed, obesity is characterized by enhanced macrophage recruitment and polarization to a proinflammatory M1 profile (1.Hotamisligil G.S. Inflammation and metabolic disorders.Nature. 2006; 444: 860-867Crossref PubMed Scopus (6276) Google Scholar), while the opposite, i.e., caloric restriction, reduces adipose tissue mass and macrophage infiltration and induces leukocyte polarization to a M2 anti-inflammatory profile (13.Morris D.L. Singer K. Lumeng C.N. Adipose tissue macrophages: phenotypic plasticity and diversity in lean and obese states.Curr. Opin. Clin. Nutr. Metab. Care. 2011; 14: 341-346Crossref PubMed Scopus (186) Google Scholar). In addition to growth factors and amino acids, mTORC1 is also activated by proinflammatory molecules, such as LPS and cytokines, via TLR4 signaling through either IKK (14.Lee D.F. Kuo H.P. Chen C.T. Hsu J.M. Chou C.K. Wei Y. Sun H.L. Li L.Y. Ping B. Huang W.C. et al.IKK beta suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway.Cell. 2007; 130: 440-455Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar) or phosphoinositide 3 kinase (PI3K)-mTOR complex 2 (mTORC2)-Akt (15.Martin M. Schifferle R.E. Cuesta N. Vogel S.N. Katz J. Michalek S.M. Role of the phosphatidylinositol 3 kinase-Akt pathway in the regulation of IL-10 and IL-12 by Porphyromonas gingivalis lipopolysaccharide.J. Immunol. 2003; 171: 717-725Crossref PubMed Scopus (221) Google Scholar, 16.Festuccia W.T. Pouliot P. Bakan I. Sabatini D.M. Laplante M. Myeloid-specific Rictor deletion induces M1 macrophage polarization and potentiates in vivo pro-inflammatory response to lipopolysaccharide.PLoS One. 2014; 9: e95432Crossref PubMed Scopus (84) Google Scholar), as well as by anti-inflammatory IL-4 and IL-13 (14.Lee D.F. Kuo H.P. Chen C.T. Hsu J.M. Chou C.K. Wei Y. Sun H.L. Li L.Y. Ping B. Huang W.C. et al.IKK beta suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway.Cell. 2007; 130: 440-455Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar), being therefore an important regulator of innate and adaptive immune responses (17.Weichhart T. Hengstschläger M. Linke M. Regulation of innate immune cell function by mTOR.Nat. Rev. Immunol. 2015; 15: 599-614Crossref PubMed Scopus (478) Google Scholar). In spite of its activation in "inflamed" fat in obesity, little is known about the role of mTORC1 in the regulation of adipose tissue inflammation. Indeed, pharmacological mTOR inhibition with rapamycin was shown to exacerbate obesity-associated adipose tissue inflammation by increasing tissue infiltration of proinflammatory M1 macrophages, as well as content of TNF-α, IL-6, and monocyte chemoattractant protein (MCP)1 (18.Paschoal V.A. Amano M.T. Belchior T. Magdalon J. Chimin P. Andrade M.L. Ortiz-Silva M. Castro É. Yamashita A.S. Rosa Neto J.C. et al.mTORC1 inhibition with rapamycin exacerbates adipose tissue inflammation in obese mice and dissociates macrophage phenotype from function.Immunobiology. 2017; 222: 261-271Crossref PubMed Scopus (37) Google Scholar, 19.Makki K. Taront S. Molendi-Coste O. Bouchaert E. Neve B. Eury E. Lobbens S. Labalette M. Duez H. Staels B. et al.Beneficial metabolic effects of rapamycin are associated with enhanced regulatory cells in diet-induced obese mice.PLoS One. 2014; 9: e92684Crossref PubMed Scopus (49) Google Scholar). Because of the systemic nature of rapamycin, partial inhibitory activity toward mTORC1, and off-target to mTORC2 (8.Laplante M. Sabatini D.M. mTOR signaling in growth control and disease.Cell. 2012; 149: 274-293Abstract Full Text Full Text PDF PubMed Scopus (6164) Google Scholar), it was impossible in these studies to delineate the contribution of adipocyte mTOR complexes in the regulation of adipose tissue inflammation. Therefore, we investigated, herein, the effects of adipocyte-specific raptor (mTORC1 deficiency) and rictor (mTORC2 deficiency) deletions on adipose tissue inflammation and glucose homeostasis in mice fed with a chow or high-fat diet (HFD). Animal experimental procedures were approved by the Animal Care Committee of the Institute of Biomedical Sciences, University of Sao Paulo, Brazil (098/2010 and 093/2012, CEUA). Mice were obtained from the Jackson Laboratory (Bar Harbor, ME) on a C57BL6/J background and kept at 22 ± 1°C, 12:12 h light-dark cycle, with free access to tap water and food ad libitum. Adipocyte raptor or rictor deletion was produced by crossing raptorLox/Lox (B6.Cg-Rptortm1.1Dmsa/J) and rictorLox/Lox (rictortm1.1Klg/SjmJ) mice with adiponectin-cre mice [B6;FVB-Tg(Adipoq-cre)1Evdr/J]. Heterozygous raptorLox/WT;adiponectin-cre and rictorLox/WT;adiponectin-cre offspring were then crossed with raptorLox/Lox and rictorLox/Lox mice to obtain raptorLox/Lox;adiponectin-cre and rictorLox/Lox;adiponectin-cre (henceforth referred to as RapKO and RicKO, respectively) and their littermates raptorLox/Lox and rictorLox/Lox (henceforth referred to as RapWT and RicWT, respectively) mice. Mice were fed with a standard chow diet (NUVILAB CR-1-Sogorb Inc., São Paulo, Brazil; 63% carbohydrates, 25% protein, and 12% fat; percent kilocalories) or a HFD (20% carbohydrates, 20% protein, 60% fat; percent kilocalories) for 8 weeks and evaluated for body weight and food intake weekly. After 8 weeks, 6 h-fasted mice were euthanized for tissue and blood harvesting. Cohorts of chow-fed RapWT and RapKO mice were treated with either vehicle or the antioxidant, N-acetylcysteine (NAC; 300 mg/kg; Sigma) or the antibiotic and serine palmitoyltransferase long chain (SPTLC) inhibitor, myriocin (MYR; 0.5 mg/kg; Sigma) once daily by gavage during 7 days. Doses of NAC and MYR were previously described (20.Ivanovski O. Szumilak D. Nguyen-Khoa T. Ruellan N. Phan O. Lacour B. Descamps-Latscha B. Drüeke T.B. Massy Z.A. The antioxidant N-acetylcysteine prevents accelerated atherosclerosis in uremic apolipoprotein E knockout mice.Kidney Int. 2005; 67: 2288-2294Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 21.Ussher J.R. Koves T.R. Cadete V.J. Zhang L. Jaswal J.S. Swyrd S.J. Lopaschuk D.G. Proctor S.D. Keung W. Muoio D.M. et al.Inhibition of de novo ceramide synthesis reverses diet-induced insulin resistance and enhances whole-body oxygen consumption.Diabetes. 2010; 59: 2453-2464Crossref PubMed Scopus (258) Google Scholar). A cohort of HFD-fed RapWT and RapKO mice was also treated with the PPARγ agonist, rosiglitazone (RSG; 30 mg/kg/day), as an admixture in the diet for 8 weeks (22.Festuccia W.T. Blanchard P.G. Belchior T. Chimin P. Paschoal V.A. Magdalon J. Hirabara S.M. Simões D. St-Pierre P. Carpinelli A. et al.PPARγ activation attenuates glucose intolerance induced by mTOR inhibition with rapamycin in rats.Am. J. Physiol. Endocrinol. Metab. 2014; 306: E1046-E1054Crossref PubMed Scopus (38) Google Scholar). Serum insulin was measured by ELISA following supplier recommendations (Millipore, Billerica, MA). Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were performed in 6 h-fasted mice injected intraperitoneally with either glucose (1 g/kg) or insulin (0.75 U/kg), respectively. Tail-vein blood glycemia was determined before and 15, 30, 45, 60, and 90 min after glucose injection or 5, 10, 15, 20, and 30 min after insulin injection using a OneTouch Johnson & Johnson glucometer. Plasma glucose disappearance rate (kITT, percent per minute) in the ITT was calculated as before (22.Festuccia W.T. Blanchard P.G. Belchior T. Chimin P. Paschoal V.A. Magdalon J. Hirabara S.M. Simões D. St-Pierre P. Carpinelli A. et al.PPARγ activation attenuates glucose intolerance induced by mTOR inhibition with rapamycin in rats.Am. J. Physiol. Endocrinol. Metab. 2014; 306: E1046-E1054Crossref PubMed Scopus (38) Google Scholar). Epididymal adipose tissue was digested with collagenase type II (1 mg/ml; Sigma-Aldrich) in Krebs-HEPES buffer containing BSA (1%) and glucose (2 mmol/l), pH 7.4 at 37°C. After filtering, the cell suspension was centrifuged (600 g, 10 min) for isolation of floating mature adipocytes and stromal-vascular cells (pellet) for PCR and flow cytometry analysis. Stromal-vascular cells (2 × 106 cells) were incubated with red blood cell lysis buffer (5 min), centrifuged, resuspended in Fc block, and analyzed for leukocyte content in a FACSCanto II, essentially as previously described (18.Paschoal V.A. Amano M.T. Belchior T. Magdalon J. Chimin P. Andrade M.L. Ortiz-Silva M. Castro É. Yamashita A.S. Rosa Neto J.C. et al.mTORC1 inhibition with rapamycin exacerbates adipose tissue inflammation in obese mice and dissociates macrophage phenotype from function.Immunobiology. 2017; 222: 261-271Crossref PubMed Scopus (37) Google Scholar). Ten thousand events per sample were acquired with a Diva-Software™ and analyzed with FlowJo 10.0.7. Antibodies used in fluorescence-activated cell sorting (FACS) analysis are detailed in supplemental Table S1. Adipose tissue, mature adipocytes, and stromal-vascular cell total RNA were extracted with Trizol (Invitrogen Life Technologies), reverse transcribed to cDNA, and evaluated by quantitative real-time PCR using a Rotor Gene (Qiagen) and SYBR Green as fluorescent dye, as previously described (16.Festuccia W.T. Pouliot P. Bakan I. Sabatini D.M. Laplante M. Myeloid-specific Rictor deletion induces M1 macrophage polarization and potentiates in vivo pro-inflammatory response to lipopolysaccharide.PLoS One. 2014; 9: e95432Crossref PubMed Scopus (84) Google Scholar). Data are expressed as the ratio of target and reference genes (36B4 and HPRT1), which were not significantly affected by raptor or rictor deletion. Primer sequences are detailed on supplemental Table S2. Protein extracts from epididymal fat pad were resolved on polyacrylamide gels, transferred to PVDF membranes, blocked with 5% milk, and incubated with primary and secondary antibodies, as previously described (9.Magdalon J. Chimin P. Belchior T. Neves R.X. Vieira-Lara M.A. Andrade M.L. Farias T.S. Bolsoni-Lopes A. Paschoal V.A. Yamashita A.S. et al.Constitutive adipocyte mTORC1 activation enhances mitochondrial activity and reduces visceral adiposity in mice.Biochim. Biophys. Acta. 2016; 1861: 430-438Crossref PubMed Scopus (30) Google Scholar). Primary antibodies are detailed in supplemental Table S3. Membranes were developed using the ECL substrate (GE Healthcare Life Sciences). Densitometric analyses were performed with ImageJ (National Institutes of Health). Epididymal adipose tissue was fixed in 4% formaldehyde and embedded in paraffin, cross-sectioned (5 μm), deparaffinized, and stained with hematoxylin-eosin. Crown-like structures (CLSs) were identified as single adipocytes surrounded by macrophages, counted along with total adipocyte number, and expressed as number per 1,000 adipocytes, essentially as previously described (23.Murano I. Barbatelli G. Parisani V. Latini C. Muzzonigro G. Castellucci M. Cinti S. Dead adipocytes, detected as crown-like structures, are prevalent in visceral fat depots of genetically obese mice.J. Lipid Res. 2008; 49: 1562-1568Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Adipose tissue malondialdehyde (MDA) content was measured with a reversed-phase HPLC (Agilent Technologies 1200 series; Santa Clara, CA) after thiobarbituric acid derivatization, as described (24.Hong Y.L. Yeh S.L. Chang C.Y. Hu M.L. Total plasma malondialdehyde levels in 16 Taiwanese college students determined by various thiobarbituric acid tests and an improved high-performance liquid chromatography-based method.Clin. Biochem. 2000; 33: 619-625Crossref PubMed Scopus (117) Google Scholar). Fluorometric detection at excitation and emission wavelengths of 515 and 543 nm, respectively, was used to quantify MDA using a standard regression prepared with 1,1,3,3-tetraethoxypropane (0.5–15.0 μM). Catalase (CAT) activity was measured as previously described (25.Aebi H. Catalase in vitro.Methods Enzymol. 1984; 105: 121-126Crossref PubMed Scopus (18599) Google Scholar). Briefly, 30 mg of tissue were homogenized in 50 mM phosphate buffer and centrifuged at 6,000 g for 10 min. The supernatant was incubated with 30 mM hydrogen peroxide for about 30 s at 20°C. The absorbance was set at 240 nm. Protein extracts from epididymal adipose tissue, prepared as previously described (9.Magdalon J. Chimin P. Belchior T. Neves R.X. Vieira-Lara M.A. Andrade M.L. Farias T.S. Bolsoni-Lopes A. Paschoal V.A. Yamashita A.S. et al.Constitutive adipocyte mTORC1 activation enhances mitochondrial activity and reduces visceral adiposity in mice.Biochim. Biophys. Acta. 2016; 1861: 430-438Crossref PubMed Scopus (30) Google Scholar), were evaluated for IL-1β content by ELISA (eBioscience, San Diego, CA), following the manufacturer'ns recommendations. Adipose tissue insulin-stimulated glucose uptake and fatty acid profile were evaluated essentially as previously described (9.Magdalon J. Chimin P. Belchior T. Neves R.X. Vieira-Lara M.A. Andrade M.L. Farias T.S. Bolsoni-Lopes A. Paschoal V.A. Yamashita A.S. et al.Constitutive adipocyte mTORC1 activation enhances mitochondrial activity and reduces visceral adiposity in mice.Biochim. Biophys. Acta. 2016; 1861: 430-438Crossref PubMed Scopus (30) Google Scholar, 26.Belchior T. Paschoal V.A. Magdalon J. Chimin P. Farias T.M. Chaves-Filho A.B. Gorjão R. St-Pierre P. Miyamoto S. Kang J.X. et al.Omega-3 fatty acids protect from diet-induced obesity, glucose intolerance, and adipose tissue inflammation through PPARγ-dependent and PPARγ-independent actions.Mol. Nutr. Food Res. 2015; 59: 957-967Crossref PubMed Scopus (43) Google Scholar). For ceramide quantification, briefly, adipose tissue was homogenized in 10 mM sodium phosphate buffer (pH 7.4) containing 100 μM deferoxamine mesylate and the internal standard, N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine (SM d18:1/17:0). Lipids were extracted with methyl-tert-butyl ether/methanol. The organic phase containing lipids was dried under nitrogen stream, suspended in isopropanol, and analyzed in an ultra (U)HPLC (Nexera; Shimadzu, Kyoto, Japan) coupled to an electrospray ionization TOF mass spectrometer (Triple TOF 6600; Sciex, Framingham, MA). Lipids were separated on a UPLC reversed-phase column (CORTECS® C18 column, 1.6 μm, 2.1 mm internal diameter × 100 mm). Lipid molecular species were identified with an in-house Excel-based macro based on the MS and MS/MS fragmentation pattern observed with PeakView®. Quantification was performed with MultiQuant®, where peak areas of precursor ions were normalized to those of the internal standards. Differences between genotypes, treatments, and their combination were evaluated by Student'ns unpaired t-tests or multifactorial ANOVA followed by Newman-Keuls test, when appropriate. Data were assessed for sphericity using Mauchly'ns test, and whenever the test was violated, technical correction through the Greenhouse-Geisser test was performed. The statistical level of significance was set at P < 0.05. Data were analyzed using Graph Prism® (GraphPad Software Inc., San Diego, CA). Results are presented as mean ± SEM. We have previously shown that pharmacological mTORC1 inhibition with rapamycin exacerbates obesity-associated glucose intolerance and adipose tissue inflammation (18.Paschoal V.A. Amano M.T. Belchior T. Magdalon J. Chimin P. Andrade M.L. Ortiz-Silva M. Castro É. Yamashita A.S. Rosa Neto J.C. et al.mTORC1 inhibition with rapamycin exacerbates adipose tissue inflammation in obese mice and dissociates macrophage phenotype from function.Immunobiology. 2017; 222: 261-271Crossref PubMed Scopus (37) Google Scholar). In an attempt to investigate whether adipocyte mTORC1 deficiency would mimic the exacerbation of fat tissue inflammation induced by rapamycin, mice with raptor deletion exclusively in adipocytes (RapKO) and littermate controls (RapWT) were fed with a chow diet or HFD for 8 weeks and evaluated for several parameters. As depicted in Fig. 1A, RapKO mice had reduced adipose tissue protein content of raptor, total and phosphorylated (p)S6 and 4E binding protein, and increased pSer473Akt, confirming the impairment in mTORC1 activity induced by raptor deletion. Confirming previous studies (10.Polak P. Cybulski N. Feige J.N. Auwerx J. Rüegg M.A. Hall M.N. Adipose-specific knockout of raptor results in lean mice with enhanced mitochondrial respiration.Cell Metab. 2008; 8: 399-410Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 11.Lee P.L. Tang Y. Li H. Guertin D.A. Raptor/mTORC1 loss in adipocytes causes progressive lipodystrophy and fatty liver disease.Mol. Metab. 2016; 5: 422-432Crossref PubMed Scopus (69) Google Scholar), RapKO mice fed with a chow diet had reduced body weight gain and masses of retroperitoneal and epididymal fat depots and were completely protected from the increase in adiposity induced by HFD intake (Fig. 1B–D). This reduced adiposity was associated with a reduction in adipocyte diameter and mRNA levels of important lipogenic enzymes, CD36, LPL, and FABP4 (supplemental Fig. S1A–C). It is noteworthy that HFD feeding increased body weight gain in RapKO mice, which seemed to be due to exacerbated hepatic lipid accumulation (Fig. 1E, F) as the result of the inability of adipose tissue to store lipids. Along with hepatic steatosis and independently of diet, RapKO mice were insulin resistant, as evidenced by the higher fasting hyperinsulinemia, insulin intolerance, and liver mRNA levels of the gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase) (Fig. 1H, K–N). No changes, however, were seen in fasting glycemia or glucose tolerance between RapKO and RapWT mice (Fig. 1G, I, J). Interestingly, despite the reduced body weight and adiposity, adipocyte mTORC1 deficiency induced adipose tissue inflammation in chow-fed mice, as evidenced by the increased percentage of resident M1 (F4/80+ CD11b+ CD86+) and M2 (F4/80+ CD11b+ CD206+) macrophages, neutrophils (Ly6G+) and B lymphocytes (CD19+), CLS density, and mRNA levels of the macrophage markers, F4/80 (total), CD86 (M1), and CD206 (M2), and the cytokines, MCP1, IL-6, and TNF-α (Fig. 2A–H). The adipose tissue percentage of dendritic cells and T lymphocytes was not altered by raptor deletion (data not shown). In addition, adipocyte mTORC1 deficiency significantly increased adipose tissue mRNA levels of inflammasome components, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC/PyCARD), and IL-1β, and protein content of pro-IL-1β and IL-1β in chow-fed mice (Fig. 2H–J). Interestingly, raptor deletion promoted inflammasome activation in adipocytes, but not in stromal vascular cells, as evidenced by the increased mRNA levels of NLRP3, DUSP-6, and IL-1β exclusively in adipocytes (supplemental Fig. S1D, E). Of note, similarly to raptor deletion, chronic rapamycin treatment significantly increased adipose tissue mRNA levels of the inflammasome components, NLRP3, ASC/PyCARD, DUSP-6, and IL-1β in HFD-fed mice without affecting body weight gain or adiposity (data not shown). Altogether, these findings indicate that adipocyte mTORC1 deficiency, despite reducing adipose tissue mass and independently of diet, promotes adipose tissue inflammation and adipocyte NLRP3-inflammasome activation. We next investigated to determine whether adipocyte deficiency of mTORC2, an upstream regulator of mTORC1 activity that is also activ
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