Ambar Grijalva, Xiaoyuan Xu, and Anthony W. Ferrante Jr.

Autophagy Is Dispensable forMacrophage-Mediated LipidHomeostasis in Adipose TissueDiabetes 2016;65:967–980 | DOI: 10.2337/db15-1219

Adipose tissue (AT) macrophages (ATMs) contribute toobesity-induced inflammation and metabolic dysfunction,but also play critical roles in maintaining tissue homeo-stasis. ATMs catabolize lipid in a lysosomal-dependentmanner required for the maintenance of AT; deficiency inlysosomal acid lipase (Lipa), the enzyme required for lyso-some lipid catabolism, leads to AT atrophy and severehepatic steatosis, phenotypes rescued by macrophage-specific expression of Lipa. Autophagy delivers cellularproducts, including lipid droplets, to lysosomes. Giventhat obesity increases autophagy in AT and contributesto lipid catabolism in other cells, it was proposed thatautophagy delivers lipid to lysosomes in ATMs and is re-quired for AT homeostasis. We found that obesity doesincrease autophagy in ATMs. However, genetic or phar-macological inhibition of autophagy does not alter thelipid balance of ATMs in vitro or in vivo. In contrast tothe deficiency of lysosomal lipid hydrolysis, the ablationof autophagy in macrophages does not lead to AT atrophyor alter metabolic phenotypes in lean or obese animals.Although the lysosomal catabolism of lipid is necessaryfor normal ATM function and AT homeostasis, delivery oflipid to lysosomes is not autophagy dependent andstrongly suggests the existence of another lipid deliverypathway critical to lysosome triglyceride hydrolysis inATMs.

Interest in adipose tissue (AT) macrophages (ATMs)developed about a decade ago when it was discoveredthat obesity increases ATM populations (1,2). Sincethen, much work (3–11) has focused on the inflam-matory functions of ATMs and their contribution toobesity-induced pathology. More recently, studies have

uncovered noninflammatory, adaptive, and homeostaticroles for ATMs, including their role in local lipid catab-olism and buffering. With the onset of obesity, ATMsaccumulate lipid and activate a catabolic program thatis lysosome dependent (12–16). In mice and humans,deficiency of lysosomal acid lipase (Lipa), which encodesthe lysosomal enzyme required for hydrolysis of tri-glycerides (TGs) and cholesterol esters, leads to ATatrophy and severe hepatic steatosis (17,18). The ex-pression of Lipa specifically by macrophages preventsthe complications of Lipa deficiency in mice, revealing acritical need for lipid metabolism by macrophages inAT homeostasis (17,19–21).

In most cells, including macrophages, neutral lipidsaccumulate in lipid droplets (LDs), specialized organ-elles that store and release lipid through classic neutrallipolysis (22). However, recent studies (23–25) revealedthat lipids in LDs also undergo hydrolysis in lysosomesvia autophagy, providing a pathway for lysosome lipiddelivery in ATMs. Indeed, foam cells, the lipid-laden mac-rophages of atherosclerotic plaques, catabolize lipids inpart through autophagy, and inhibition of autophagyleads to the accumulation of lipids (24). Autophagy isan evolutionarily conserved process by which single mol-ecules, macromolecules, and organelles are delivered tolysosomes for degradation (26–29). The metabolic stateof a cell or tissue profoundly regulates autophagy, withfasting or nutrient deprivation typically activating auto-phagy. The effects of overnutrition are more complex. Inliver and foam cells, obesity impairs autophagy, whereas,conversely, when whole AT is analyzed, obesity has beenfound to activate autophagy (26–35). We and others hadhypothesized, therefore, that autophagy delivers lipids to

Department of Medicine, The Naomi Berrie Diabetes Center, Columbia University,New York, NY

Corresponding author: Anthony W. Ferrante Jr., awf7@columbia.edu.

Received 1 September 2015 and accepted 15 January 2016.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1219/-/DC1.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

Diabetes Volume 65, April 2016 967

OBESITYSTUDIES

lysosomes in ATMs of obese animals and that impairmentof autophagy would phenocopy the effects of Lipa defi-ciency (i.e., lipoatrophy and hepatic steatosis). Using bothin vivo and in vitro systems, we found that excess lipidsand obesity activate autophagy in ATMs, but, surprisingly,impairing autophagy genetically or pharmacologicallydoes not alter the lipid content of ATMs in lean or obeseanimals, does not alter AT mass, and does not cause he-patic steatosis or impair systemic metabolism.

RESEARCH DESIGN AND METHODS

Animals and Animal CareMale C57BL/6J and B6.V-Lep/obJ mice were obtainedfrom The Jackson Laboratory (Bar Harbor, ME) at 10–12weeks of age. To generate mice with myeloid-specificdeletion of Atg7 (MacAtg7 KO mice), we crossed LysMCreB6.129P2-Lyz2tm1(cre)Ifo/J (The Jackson Laboratory) andC57BL/6 Atg7F/F mice (a gift from Masaaki Komatsu, TokyoMetropolitan Institute of Medical Science, Tokyo, Japan),subsequently generating MacAtg7 KO (Atg7F/F; LysMCre) andlittermate controls (Atg7F/F) (36,37). Mouse genotypes weredetermined using PCR amplification of DNA from tail lysateor blood immune cells (36). Mice were housed in a pathogen-free barrier facility, in ventilated cages with free access toautoclaved water, and were fed either a low-fat pellet diet(5% calories from fat) (PicoLab Rodent Diet 20; Purina MillsInc) or high-fat diet (60% calories from fat) (D12492; Re-search Diets). Mice were maintained on a 12-h light/darkcycle and housed with three to five male mice per cage.Metabolic measurements were made beginning at 10 weeksof age until sacrifice at 19 weeks of age (lean mice) or26 weeks of age (DIO mice). The Columbia UniversityInternational Animal Care and Use Committee ap-proved all procedures.

Isolation and Culturing of Stromal Vascular CellsFollowing CO2 asphyxiation and cervical dislocation, peri-gonadal AT (PGAT) was isolated using sterile techniques.PGAT was placed in FACS buffer (PBS; GIBCO), 0.2% BSA(Sigma-Aldrich), and 5 mmol/L EDTA (Sigma-Aldrich), andwas minced into fine (,10 mg) pieces. Samples werecentrifuged at 500g for 5 min. PGAT was then digestedin DMEM (Invitrogen) with 10 mg/mL BSA and digestiveenzymes (0.14 units/mL Liberase TM (Roche Applied Sci-ence) and 50 units/mL DNase I (Sigma-Aldrich) for 30 minat 37°C at 185 rpm on an orbital shaker. The digestedmaterial was passed through a sterile 250-mm nylonmesh (Sefar Filtration Inc.) and centrifuged for 5 min at500g. The stromal vascular cells (SVCs) comprising thepellet were washed with FACS buffer and centrifuged at500g for 5 min. For flow cytometry, SVCs were resus-pended in erythrocyte lysis buffer (BD Biosciences). Forimmunofluorescence staining, SVCs were resuspended inculture medium (DMEM, 10% FBS [Invitrogen], and 1%penicillin-streptomycin [Invitrogen]) at 500,000 cells/mL,and were cultured overnight at 37°C, with 5% CO2 in four-well chamber slides (LabTek). For Western blot analysis,

cultured SVCs were treated with 20 mmol/L chloroquine(CQ; Sigma-Aldrich) or deionized H2O for 16 h, collectedusing 0.02% EDTA (Sigma-Aldrich), and resuspended inTissue Extraction Reagent I (Invitrogen) with a proteaseinhibitor cocktail (Sigma-Aldrich).

Differentiation of Bone Marrow–Derived ATMs andBone Marrow–Derived Foam CellsBone marrow (BM)-derived ATMs (BM-ATMs) were dif-ferentiated as previously described (13). Briefly, BM cellswere collected from femurs of 6- to 10-week-old control(Atg7F/F), MacAtg7 KO (LysMCre+Atg7F/F), control (Lipa+/+),or Lipa knockout (KO; Lipa2/2) mice by flushing with BMculture medium (minimum essential medium a [Invitro-gen], 10% FBS, 1% nonessential amino acids [Invitrogen],1% penicillin-streptomycin) (18). Cells were plated using50–60 3 106 cells/100 mL in a 175-mL tissue cultureflask at 37°C in 5% CO2. On the second day, nonadherentcells were collected, centrifuged at 500g for 5 min, andplated at 1.5 3 106 cells/2 mL in BM culture mediumsupplemented with 30 ng/mL human macrophage colonystimulating factor (M-CSF; R&D Systems). After 3 d ofculture with M-CSF, adherent cells were differentiatedinto either BM-macrophages (BM-Macs), BM-ATMs, orBM-foam cells. BM-Macs were generated by addition offresh M-CSF supplemented BM and continued culture forfive days. To generate BM-ATMs, on day three of culture100 mg of PGAT was placed in porous tissue inserts (BDBiosciences) and added to adherent cells wells. To gener-ate BM-foam cells, 150 mg/mL acetylated LDL (BiomedicalTechnologies, Inc.) was added to BM-Macs for 24 h(24,31). Lysosomal function was inhibited in cells by add-ing 20 mmol/L CQ for 16 h. On the eighth day, cells werecollected using 0.02% EDTA (Sigma-Aldrich) for flowcytometry or protein isolation, or were used for immuno-fluorescence staining.

Flow CytometrySVCs were resuspended in erythrocyte lysis buffer in-cubated at room temperature for 3 min and centrifugedat 500g for 5 min. Erythrocyte-free SVCs or collected BMcells were resuspended in FACS buffer at 73 106 cells/mLwith 2% Fc Block (BD Biosciences) for 30 min at 4°C.Fluorophore-conjugated antibodies were added for 30 minat 4°C with rotation, as follows: anti-CD45.2-Percp-Cy5.5(1:100, v/v) immune cell marker (BD Biosciences), anti-F4/80-APC (1:20, v/v) macrophage marker (AbD Serotec),anti-CD11b (1:100, v/v) macrophage marker (Invitrogen),anti-CD11c-PE-TR (1:100, v/v), ATM marker (Invitrogen),and 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503)–FITC (1:300 v/v) lipidmarker (Invitrogen). Cells were resuspended in FACS bufferwith DAPI (Invitrogen) and analyzed using an LSRII FlowCytometer (Becton Dickson). Data analysis was performedwith FlowJo software.

Immunofluorescence MicroscopyCultured cells were stained with 1:300 (v/v) BODIPYfor neutral lipid and 5 nmol/L LysoTracker (Invitrogen)

968 Autophagy in Adipose Tissue Macrophages Diabetes Volume 65, April 2016

Figure 1—ATMs accumulate and catabolize neutral lipid in a Lipa-dependent manner. A: PGAT SVCs from lean (Lep+/+) and obese (Lepob/ob)mice were isolated. SVCs were stained using CD45 antibody, an immune cell marker; F4/80 antibody, a macrophage marker; and BODIPY,a neutral lipid dye; and were analyzed using flow cytometry. Representative flow cytometry histogram of BODIPY MFI in lean and obeseCD45+F4/80+ (ATMs). Black line = lean ATMs, dashed line = obese ATMs. B: Quantification of relative BODIPY MFI in CD45+F4/80+ cells(ATMs) from lean and obese mice. White bar = lean ATMs, black bar = obese ATMs. n = 5/group. Values are reported as the mean 6SEM. **P < 0.01. C: PGAT SVCs from lean (Lep+/+) and obese (Lepob/ob) mice were cultured overnight, and immunofluorescencestaining was performed by incubating SVCs with BODIPY. SVCs were then fixed, blocked, and stained with anti-F4/80 antibody tohighlight ATMs. Scale bar = 10 mm. D: Quantification of relative BODIPY MFI in macrophage cells was determined using Nikon NIS-Elements Software. White bar = lean ATMs, black bar = obese ATMs. n = 6/group. Values are reported as the mean 6 SEM. **P < 0.01.E: SVCs from lean and obese mice were cultured for 4 or 48 h, fixed, stained with Oil Red O for neutral lipids, and counterstained withhematoxylin-eosin, all at 603 magnification. F: BM cells were isolated from control (Lipa+/+) and Lipa KO (Lipa2/2) mice and were

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for lysosomes for 30 min, then were fixed with zincformaldehyde (Z-Fix; Anatech Ltd.) for 10 min. Forintracellular staining, cells were permeabilized using ice-cold methanol (Fisher Scientific) at 220°C for 10 min.Cells were blocked in blocking buffer (PBS, 10% goat se-rum; Sigma-Aldrich) for 1 h at room temperature. Primaryantibodies were diluted in antibody dilution buffer(Cell Signaling Technology) containing PBS, 1% BSA(Sigma-Aldrich), and 0.3% Triton X-100 (Sigma-Aldrich),and were incubated overnight at 4°C. The followingantibodies were used: anti-F4/80 (1 mg/mL), macro-phage antibody (Invitrogen), anti-LC3 (1:250, v/v), anautophagosome marker (Cell Signaling Technology). Onthe following day, cells were incubated with secondary an-tibodies for 2 h at room temperature, as follows: 1:400 (v/v)Cy5 or Cy3 (Invitrogen). Samples were washed and incu-bated with 1:4,000 (v/v) DAPI for 10 min to visualize thenuclei. Glass coverslips were mounted using Fluoro-GelMounting Solution (Electron Microscopy Sciences). Slideswere imaged using Nikon A1R MP Confocal Microscope.The mean fluorescence intensity (MFI) of BODIPY andLC3 was measured within F4/80+ cells. Background (av-erage fluorescent intensity outside of cells) was subtractedfrom fluorescence signals, and the MFI per macrophagewas measured. MFIs were calculated from individual im-ages of all images from one well. Data were analyzed onNikon NIS Elements imaging software.

Immunohistochemistry MicroscopyTissues were fixed for 48 h in Z-fix, washed with 70% ethanolfor 24 h, then embedded in paraffin. The 5-mm sections werecut at 50-mm intervals and mounted on glass slides, depar-affinized in xylene, and counterstained with hematoxylin-eosin. To stain neutral lipids, SVCs were fixed with Z-fix for10 min and stained with Oil Red O solution (Sigma-Aldrich)for 10 min, washed three times in deionized water, andcounterstained with hematoxylin-eosin for nuclear staining.

Western BlotSVCs and BM cells resuspended in tissue extraction reagentI (Invitrogen) with a protease inhibitor cocktail (Sigma-Aldrich) were homogenized and sonicated. Protein concen-tration was measured using Bio-Rad Protein Assay. Twentyto fifty micrograms of protein was denatured at 96°C for5 min in 43 protein sample buffer (200 mmol/L Tris 6.8,8% SDS, 0.4% bromophenol blue, 40% glycerol, and 5%b-mercaptoethanol) and loaded on 14% SDS-polyacrylamidegels (Invitrogen). Samples were transferred on nitrocellu-lose (Thermo Scientific) or polyvinylidene fluoride (FisherScientific) using either a semidry or wet transfer system(Bio-Rad). Membranes were counterstained with Ponceaustain, washed with Tris-buffered saline with Tween-20

(TBST), blocked with a 5% nonfat milk solution, and incu-bated with primary antibodies (anti-LC3 [Cell Signaling Tech-nology], anti-Atg7 [Cell Signaling Technology], anti-Atgl [CellSignaling Technology], and anti–b-actin [Sigma-Aldrich]) at4°C overnight. Membranes were washed with TBST, incubatedwith secondary antibodies, and developed using an enhancedchemiluminescence detection system (GE Healthcare Life Sci-ences) or the fluorescence Odyssey CLx Imaging System(LI-COR). Band intensities were determined using image anal-ysis (Quantity One; Bio-Rad) and were normalized to b-actin.

Metabolic StudiesSerum metabolites and hormones were measured after a 6-hfast. Blood was collected for glucose and serum analysisthrough a submandibular bleed. Glucose was measuredusing a Freestyle Blood Glucometer (Abbott Laboratories).Serum measurements included insulin (Mouse Ultrasensi-tive Insulin ELISA; ALPCO), TGs (Infinity TriglyceridesReagent; Thermo Scientific), and nonesterified fatty acids(NEFAs; NEFA-HR; Wako Diagnostics). Body compositionmeasurements were made with a minspec TD NMR analyzer(Bruker). Mice were fasted for 24 h before sacrifice. Tissueswere stored in280°C for later analysis, fixed for histologicalsectioning, or digested for SVC isolation.

Statistical AnalysisData are presented as the mean 6 SEM. Differences weredeemed significant if P values were ,0.05 or correctedP values were ,0.001. Sample comparison statistics weredetermined using a Student t test (two-tailed distribution,two-sample unequal variance) with a Bonferroni correc-tion to analyze metabolic studies. All data were analyzedusing Microsoft Excel and GraphPad Prism.

RESULTS

ATMs uptake and catabolize lipid in a manner thatdepends on Lipa and is strongly induced by obesity. Theabsence of Lipa in macrophages leads to the accumulationof lipid in ATMs, AT atrophy, and severe hepatic steatosis(12–16). We used flow cytometry, confocal microscopy,and immunohistochemistry to assess lipid content inATMs from PGAT under various experimental conditions.Using BODIPY, a fluorescent dye that quantitativelystains neutral lipids, we found, consistent with previousreports, that the concentration of lipids in ATMs fromleptin-deficient obese (Lepob/ob) mice is higher than inATMs from lean (Lep+/+) animals (Fig. 1A–D). Ex vivo,the catabolism of neutral lipid overtime is evident inATMs from both lean and obese mice with decreases inlipid content between 4 and 48 h after isolation (Fig. 1E).

Using pharmacological inhibition of lysosomes, we pre-viously demonstrated that lipid catabolism in ATMs is

differentiated in the presence of AT to generate BM-ATMs. Immunofluorescence staining was performed using BODIPY for neutral lipids,LysoTracker for acidic compartments/lysosomes, and anti-F4/80 antibody for macrophages. Scale bar = 50 mm. G: Quantification of relativeBODIPY MFI in control (Lipa+/+) and Lipa KO (Lipa2/2) macrophage cells was determined using Nikon NIS-Elements Software. White bar =Control BM-ATMs, black bar = Lipa KO BM-ATMs. n = 10/group. Values are reported as the mean 6 SEM. *P < 0.05.

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lysosome dependent (13). To determine whether LIPA, theonly known lysosomal lipase, is required for lipid catabolismin ATMs, we differentiated BM stem cells from control(Lipa+/+) and Lipa deficient (Lipa2/2) mice in the presenceof AT to generate BM-ATMs and Lipa KO BM-ATMs (18). Asdemonstrated previously, BM-ATMs mimic in vivo obeseATMs by expressing CD11c antigen, accumulating lipids,increasing lysosomal biogenesis, and forming multinucleatedgiant cells (13). Consistent with the catabolism of lipids inATMs being Lipa dependent, the absence of Lipa increasesthe lipid content of BM-ATMs (Fig. 1F and G).

Autophagy is a process in which cellular content isdelivered to lysosomes for degradation (26,27). LDs orportions of them can be incorporated into autophagosomesand conveyed to lysosomes through macroautophagy in a

process dubbed lipophagy (25,27). Lipophagy occurs inhepatocytes, AgRP+ neurons, and foam cells, and is al-tered by obesity (23–25,30,32). Lipophagy requires theconjugation of microtubule-associated protein lightchain 3 (LC3-I) with a phosphatidylethanolamine toyield LC3-II as part of the functional complex that formsthe autophagosome membrane (38). LC3-II is a markerof autophagy and flux through autophagic pathways andcan be assessed by measuring the accumulation of LC3-IIwhen lysosome function is inhibited (i.e., the accumula-tion of LC3-II occurs because fusion of autophagosomeswith lysosomes is prevented) (38). Similar to lipid-ladenfoam cells (Supplementary Fig. 1), autophagic flux is in-creased more than threefold in BM-ATMs relative toBM-Macs (Fig. 2A and B) (24). The treatment of ATMs

Figure 2—AT-induced differentiation of ATMs increases autophagy. BM cells were isolated from mice and differentiated with M-CSF togenerate BM-Macs. BM-Macs were further differentiated with M-CSF (BM-Macs) and with AT to generate BM-ATMs. A: Immunofluorescencestaining of lipid (BODIPY), autophagy (anti-LC3 antibody), and macrophages (anti-F4/80 antibody) in BM-Macs and BM-ATMs. Scale bar =20 mm. B: Quantification of relative LC3 MFI in macrophage cells was determined using Nikon NIS-Elements Software. White bar = BM-Macs,black bar = BM-ATMs. n = 8/group. Values are reported as the mean 6 SEM. **P < 0.01. C: Representative LC3-I, LC3-II, and b-actin Westernblot for BM-ATMs and BM-ATMs treated with 20 mmol/L CQ for 16 h (BM-ATMs + CQ). D: Band intensity for LC3-II was normalized to b-actin.White bar = BM-ATMs, black bar = BM-ATMs + CQ. n = 5/group. Values are reported as the mean 6 SEM. *P < 0.05.

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Figure 3—Obesity induces autophagy in ATMs. A: PGAT SVCs were isolated from lean (Lep+/+) and obese (Lepob/ob) mice, cultured overnight,and treated with 20 mmol/L CQ or deionized H2O for 16 h. Representative Western blots for LC3-I, LC3-II, and b-actin. B: Quantification of relativeLC3-II protein levels, normalized to b-actin. White bar = lean SVCs, black bar = lean SVCs treated with CQ (lean SVC + CQ), dotted bar = obeseSVCs, horizontal striped bar = obese SVCs treated with CQ (obese SVC + CQ). n = 5/group. Values are reported as the mean6 SEM. *P< 0.05,**P < 0.01. *Untreated vs. CQ treated, +P < 0.05 lean SVC vs. obese SVC, #P < 0.05 lean SVC + CQ vs. obese SVC + CQ. C: Isolated SVCsfrom lean (Lep+/+) and obese (Lepob/ob) mice were stained with anti-LC3 (autophagy) antibody, BODIPY (neutral lipids), anti-F4/80 antibody(macrophages), and DAPI (nuclei). Scale bar = 10 mm. D: Quantification of relative LC3 MFI in macrophage cells was determined using NikonNIS-Elements Software. White bar = lean ATMs, black bar = obese ATMs. n = 6/group. Values are reported as the mean 6 SEM. *P < 0.05.

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Figure 4—Autophagy does not regulate macrophage differentiation and lipid catabolism in in vitro ATMs. BM cells were isolated fromcontrol (Atg7F/F) and MacAtg7 KO (LysMCre+Atg7F/F) mice and were differentiated with M-CSF to generate BM-Macs. BM-Macs were furtherdifferentiated with M-CSF (BM-Macs) and with AT to generate BM-ATMs. A: Flow cytometry analysis of F4/80+ CD11b+ (FB) cells in controland MacAtg7 KO BM-Macs and BM-ATMs. White bar = Control BM-Macs; black bar = MacAtg7 KO BM-Macs; dotted bar = Control BM-ATMs;horizontal striped bar = MacAtg7 KO BM-ATMs. n = 15/group. Values are reported as the mean6 SEM. B: Immunofluorescence staining wasperformed using BODIPY to quantify neutral lipid content, LC3 for autophagy, and F4/80 for macrophages in BM-ATMs. Scale bar = 50 mm.C: Quantification of relative BODIPY MFI in macrophage cells. Nikon NIS-Elements Software was used to assess data. White bar = ControlBM-Macs, black bar = MacAtg7 KO BM-Macs, dotted bar = Control BM-ATMs, horizontal striped bar = MacAtg7 KO BM-ATMs. n = 8/group.Values are reported as the mean6 SEM. **P< 0.01, compared with genotype BM-Macs. D: BM-Macs from control and MacAtg7 KO mice were

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with CQ confirms that LC3-II accumulation in these cellsis not due to a defect in autophagy machinery per se (Fig.2C and D).

These data suggest that, in vitro, AT induces autophagy inmacrophages. To determine whether obesity similarly acti-vated autophagy in primary ATMs in vivo, we isolated SVCsfrom fat of lean (Lep+/+) and obese (Lepob/ob) animals. AmongSVCs, we found that obesity increased the LC3-II content ofcells (Fig. 3A and B). Treatment with CQ further increasedLC3-II content in SVCs from obese mice compared withthose from lean mice, which is consistent with an increasein autophagic flux in primary cells (Fig. 3A and B). Usingimmunofluorescence, we found that LC3 content in bothATMs and non-ATMs cells was similar, but in obese miceLC3 specifically increased in ATMs (Fig. 3C). Consistent withour in vitro data, autophagy is increased in ATMs during thedevelopment of obesity (Fig. 3D). Fasting increases lipid ac-cumulation in ATMs and broadly activates autophagy insome key tissue. AT does not behave the same as mostorgans. Autophagy, as measured by LC3-II, was not increasedin whole AT or SVCs, suggesting that, unlike most tissues,overnutrition, not fasting, specifically activates autophagy inAT and its constituents (Supplementary Fig. 2).

The concurrent increase in lysosomal-dependent lipidcatabolism and autophagy in ATMs during the develop-ment of obesity suggested that lipid delivery to lysosomesoccurs via lipophagy. To directly assess the role of auto-phagy in ATM lipid catabolism and AT homeostasis, wedeleted Atg7, a gene required for autophagy, from myeloidcells. Using a Cre/loxP recombination system, we crossedmice carrying a floxed Atg7 allele (Atg7F/F) with mice inwhich the Cre-recombinase was inserted in the Lysozyme2 gene and expressed in myeloid cells, including macro-phages (36,37). We confirmed the deletion of Atg7 fromcirculating myeloid cells, BM-Macs, whole AT, and SVCs(Supplementary Fig. 3). Although some studies (39,40)have implicated autophagy in M-CSF–dependent monocytedifferentiation into macrophages, we found no differencein the efficiency of differentiation between control andMacAtg7 KO BM-Macs and BM-ATMs (Fig. 4A).

To determine whether lipid catabolism in ATMs isdependent upon autophagy, we studied BM-ATMs andprimary ATMs from control and MacAtg7 KO mice. Quan-tifying lipid content using a fluorescent neutral lipid dye,we found no difference in lipid content between controland MacAtg7 KO BM-ATMs (Fig. 4B and C). In contrast andconsistent with previous reports of foam cell lipid catabolism

being dependent in part on autophagy, MacAtg7 KO BM-foam cells had twice the lipid content of control BM-foamcells (Fig. 4D and E). To confirm the unexpected findingthat autophagy does not regulate lysosomal lipid metab-olism in ATMs, we pharmacologically inhibited autophagyin BM-ATMs with 3-methyladenine (3-MA), and, consis-tent with our genetic findings, 3-MA did not alter lipidcontent in BM-ATMs (Fig. 4F and G). In contrast, lysosomalinhibition with CQ did increase lipid content in control andMacAtg7 KO BM-ATMs (Supplementary Fig. 4). The data dem-onstrate that lipid catabolism in ATMs is lysosomal depen-dent but not autophagy dependent in vitro.

To study autophagy-dependent metabolism of lipids invivo, we isolated SVCs from lean and obese (high fat–fed)mice. If impairing autophagy reduces the delivery of lipidto lysosomes, then we predicted an increase in either theproportion of lipid-containing ATMs or an increase in thelipid content of each ATM. However, targeted deletion ofAtg7 did not alter the distribution or size of ATM sub-populations in MacAtg7 KO mice, in either lean or obeseanimals (Fig. 5A). Neither CD11c2ATMs (FBs), a subpop-ulation of macrophages low in lipid content, nor CD11c+

ATMs (FBCs), a subpopulation with the highest lipid con-tent, were affected by the deletion of Atg7 (Fig. 5B).

The deficiency of autophagy also did not affect the lipidcontent of individual ATMs. The FB and FBC ATMs fromcontrol and MacAtg7 KO mice contained equal amounts ofneutral lipid in both lean and obese mice (Fig. 5C and D).This was confirmed histologically (Fig. 5E). The lack ofeffect on lipid content was not due to an induction ofthe primary cytosolic lipase adipose TG lipase (ATGL/PNPLA2) (Supplementary Fig. 5) (41).

Impairing the lysosomal catabolism of lipids in mac-rophages in addition to increasing the lipid content ofATMs also leads to AT atrophy and severe hepaticsteatosis (18). However, consistent with normal ATMlipid metabolism and in stark contrast to myeloid Lipadeficiency, there was no effect of autophagy deficiencyon body weight (data not shown), body composition, fatmass distribution, or organ size (i.e., no hepatosplenome-galy) (Fig. 6A–C). The analysis of AT depots confirmedthat adipocyte size and numbers were not histologicallydifferent between control and MacAtg7 KO PGAT (Fig. 6D).Macrophage lysosome lipid catabolism is also requiredfor normal systemic lipid homeostasis (19). However, inMacAtg7 KO mice the concentrations of serum TGs and NEFAswere not different than control mice (Fig. 6E and F).

treated with 150 mg/mL acetylated low-density lipoprotein to generate BM-foam cells. Cells were stained for lipid (BODIPY), autophagy(anti-LC3 antibody), and macrophages (anti-F4/80 antibody). Scale bar = 50 mm. E: Quantification of relative BODIPY MFI in Control andMacAtg7 KO BM-foam cells. BODIPY MFI was calculated using Nikon NIS-Elements Software. White bar = Control BM-Macs, black bar =MacAtg7 KO BM-Macs, dotted bar = Control BM-foam cells, and horizontal striped bar = MacAtg7 KO BM-foam cells, n = 11/group. Values arereported as the mean6 SEM. ***P< 0.005 BM-Macs vs. BM-ATMs (control or MacAtg7 KO); +P< 0.05 control vs. MacAtg7 KO BM-foam cells.F: BM-ATMs were treated with 5 mmol/L 3-MA or vehicle for 16 h. Immunofluorescence staining using BODIPY, anti-LC3 antibody, andanti-F4/80 antibody was performed. Scale bar = 20 mm. G: Quantification of relative BODIPY MFI in 3-MA– or vehicle-treated BM-ATMswas determined using Nikon NIS-Elements Software. White bar = vehicle-treated BM-ATMs, black bar = 3-MA–treated BM-ATMs. n = 10 ton = 10/group. Values are reported as the mean 6 SEM.

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Figure 5—Autophagy does not regulate macrophage differentiation and lipid catabolism in primary ATMs. SVCs from low-fat diet–fed(Lean) and high-fat diet–fed (DIO) littermate controls (Atg7F/F) and MacAtg7 KO (LysMCre+Atg7F/F) mice were analyzed using flow cytometryfor macrophage percentages and lipid content. Flow cytometry analysis of total ATMs (CD45+F4/80+) (A) and two macrophage popula-tions, FB (CD45+F4/80+CD11b+) and FBC (CD45+F4/80+CD11b+CD11c+) cells (B), in lean and DIO control and MacAtg7 KO SVCs. Whitebar = Lean Control mice, black bar = Lean MacAtg7 KO mice, dotted bar = DIO Control mice, and horizontal striped bar = DIO MacAtg7 KO

mice. Lean Control n = 4, Lean MacAtg7 KO n = 8, DIO Control n = 4, DIO MacAtg7 KO n = 11. Values are reported as the mean 6 SEM. *P<0.05, ***P< 0.005 compared with lean genotype. C: SVCs from Lean littermate controls (Atg7F/F) and MacAtg7 KO (LysMCre+Atg7F/F) micewere analyzed using flow cytometry for lipid content (BODIPY). Representative flow cytometry histograms are shown for BODIPY

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Fasting and fed glucose and insulin values were also similarbetween control and MacAtg7 KO mice (Fig. 6G and H).

Although autophagy-dependent catabolism of lipid isnot required for ATM function and AT homeostasis in leananimals, obesity increases autophagy in ATMs. To testwhether under the increased stress of obesity AT requiresATM autophagy for homeostasis, we placed control andMacAtg7 KO mice on a high-fat diet. Control and MacAtg7 KO

mice were both comparably susceptible to diet-induced obe-sity (Fig. 7A–C). There were no differences in fat depot,liver, and spleen weights between control and MacAtg7 KO

mice (Fig. 7D). Histologically, PGAT from MacAtg7 KO micewas indistinguishable from the fat of control animals (Fig.7E). Circulating TG, NEFA, glucose, and insulin concentrationswere not different between obese control and MacAtg7 KO

mice either in the fasted or fed state (Fig. 7F–I).

DISCUSSION

With the onset of obesity, adipocytes become hypertrophicand store lipid with reduced efficiency (42). Simultaneously,AT is infiltrated by immune cells, with ATMs comprising themajority (1,2). ATMs exert inflammatory actions that cancontribute to the adverse effects of obesity on local andsystemic metabolism (3–11). However, ATMs also serveadaptive functions that include a recently identified pathwayof lysosomal lipid catabolism (12–15). The importance ofthis pathway is revealed by the following two observations:adipose atrophy and severe hepatic steatosis develop-ment in mice and people deficient in the enzyme requiredfor lysosome lipid hydrolysis (LIPA) and prevention of thephenotypes by macrophage-specific expression of LIPA(17,19–21). Efforts to understand the mechanisms bywhich macrophage-lipid catabolism contributes to AT ho-meostasis promise to provide insights into AT biology andsystemic lipid metabolism, but require defining the cellularand tissue-specific processes involved in lipid delivery tolysosomes.

Recent studies (25) have found that the delivery ofneutral lipids to lysosomes from lipophagy is critical forlipid homeostasis in hepatocytes. Ablation of Atg7 in he-patocytes increases hepatic TGs and cholesterol, and hasbeen implicated in obesity-induced hepatic steatosis (25).In AT, autophagy has been implicated in adipocyte differ-entiation with the deletion of Atg7 in adipocytes, result-ing in a reduction of white AT mass and a compensatoryincrease in brown fat and energy expenditure (43,44). A

similar lean phenotype is observed in mice lacking Atg7 inAgRP+ neurons (23). Given these findings and that macro-phage autophagy contributes to lipid catabolism in foam cellsas well as cellular differentiation and macrophage apoptosis,inflammasome activation, and clearance of dead cells/pathogens, it seemed likely that lipid delivery to lysosomesin ATMs would occur via autophagy (24,29,31,35,40,45).Furthermore, defective autophagy has been implicated inthe defective processing of excess lipid and debris by foamcells in atherosclerotic plaques (35,46).

Here in studying the delivery of lipid to lysosomes inmacrophages, we expected that autophagy would providean important transport mechanism. Instead, we foundthat, although obesity does activate autophagy in ATMs,autophagy is dispensable for lipid-dependent degradationof TGs by ATMs both in vitro and in vivo. Furthermore,unlike impairing lysosomal lipid metabolism, disablingautophagy in macrophages has no discernable effects onAT or whole-body metabolism.

These findings argue that a pathway distinct fromclassic autophagy delivers lipid to lysosomes in ATMs.Two possibilities come to mind. It is possible that ATMspossess a novel, nonclassic autophagy pathway that doesnot depend upon Atg7 and cannot be inhibited by 3-MAbut still delivers lipid from LDs to lysosomes. There havebeen reports (47) of pathways that do not require specificATGs, but even those reports suggest that chemical inhib-itors are capable of blocking delivery to lysosomes. Wehave no evidence for such a pathway, which would requireidentifying molecular components of such a process anddeveloping tools to inhibit their function.

An alternative possibility is that the lipid in ATMs is notin stored LDs, but is contained within a distinct set of lipidvesicles. The delivery of LDs or parts thereof to lysosomesdepends on autophagy because their surface is delimited bya monolayer of amphipathic molecules, which by itself isnot capable of direct fusion with lysosomes (22,48). Otherorganelles, including endosomes, possess bilayer mem-branes that permit fusion with lysosomes directly (49).Macrophages are professional phagocytes and deliver adiverse range of molecules, including lipids, to endocyticvesicles. Such vesicles can be rapidly be targeted to lyso-somes. Therefore, it seems possible that lipids in ATMsare in fact contained within non-LD vesicles. However, iflipids in ATMs are contained within such vesicles, thequestion arises as to how and in what form the lipid is

fluorescence from CD45+F4/80+ gate (total ATMs), CD45+F4/80+Cd11b+ gate (FB), CD45+F4/80+ CD11b+CD11c+ gate (FBC). Black line =Lean Control, dotted line = Lean MacAtg7 KO. Relative BODIPY MFI was quantified for all three different macrophage populations.White bar = Lean Control, black bar = Lean MacAtg7 KO. Lean Control n = 4, Lean MacAtg7 KO n = 8. Values are reported as the mean 6SEM. D: SVCs from DIO littermate controls (Atg7F/F) and Mac Atg7 KO (LysMCre+Atg7F/F) mice were analyzed, and representative flowcytometry histograms are shown for BODIPY fluorescence from CD45+F4/80+ gates (total ATMs), CD45+F4/80+Cd11b+ gates (FB),CD45+F4/80+CD11b+CD11c+ gates (FBC). Black line = DIO Control, dotted line = DIO MacAtg7 KO. Relative BODIPY MFI was quantified forall three different macrophage populations. White bar = DIO Control, black bar = DIO MacAtg7 KO. DIO Control n = 4, DIO MacAtg7 KO n = 11.Values are reported as the mean 6 SEM. E: Immunofluorescence staining using BODIPY to assess lipid content in SVCs isolated from DIOcontrol and MacAtg7 KO mice. Scale bar = 50 mm.

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Figure 6—Targeted deletion of autophagy in myeloid cells does not affect AT development or systemic metabolism in lean mice. Littermatecontrols (Atg7F/F) and MacAtg7 KO (LysMCre+Atg7F/F) mice were studied for metabolic phenotypes. A: Fat mass (grams) as the percentageof body weight (grams) (% Fat Mass). B: Lean mass (grams) as a percentage of body weight (grams) (% Lean Mass). C: Tissue weights(grams) as a percentage of body weight (grams) at 19 weeks old (% Tissue Weight). D: PGAT sections were stained with hematoxylin-eosin.Representative images at 403 magnification. Mice were bled after a 6-h fast or 8 h into the light cycle and serum metabolites weremeasured, including serum TGs (mg/dL) (E), serum NEFAs (mmol/L) (F ), serum glucose (mg/dL) (G), and serum insulin (ng/mL) (H). Whitesquare/bar = Lean Control mice, black square/bar = Lean MacAtg7 KO mice. Lean Control n = 7, Lean MacAtg7 KO n = 18. Values are reportedas the mean 6 SEM. BAT, brown AT; SCAT, subcutaneous AT.

diabetes.diabetesjournals.org Grijalva, Xu, and Ferrante 977

Figure 7—Targeted deletion of autophagy in myeloid cells does not affect AT development or systemic metabolism in obese mice. Littermatecontrols (Atg7F/F) and MacAtg7 KO (LysMCre+Atg7F/F) mice were studied for metabolic phenotypes. Beginning at 10 weeks old, mice were fed a60% high-fat diet for 16 weeks, until they were 26 weeks old. Metabolic phenotypes, including body weight (grams) (A), fat mass (grams) as apercentage of body weight (grams) (% Fat Mass) (B), lean mass (grams) as a percentage of body weight (grams) (% Lean Mass) (C), tissueweight (grams) as a percentage of body weight (grams) (D) at 26 weeks old (% Tissue weight), were assessed throughout the study. E: PGATsections were stained with hematoxylin-eosin. Representative images at 403magnification. Mice were bled after a 6-h fast or 8 h into the lightcycle, and serum was collected to measure serum metabolites, including serum TGs (mg/dL) (F), serum NEFAs (mmol/L) (G), serum glucose(mg/dL) (H), and serum insulin (ng/mL) (I). White square/bar = DIO Control mice, black square/bar = MacAtg7 KO DIO mice. DIO Control n = 4,DIO MacAtg7 KO n = 13. Values are reported as the mean 6 SEM. BAT, brown AT; SCAT, subcutaneous AT.

978 Autophagy in Adipose Tissue Macrophages Diabetes Volume 65, April 2016

taken up by ATMs. Hepatic- and intestinal-derived lipo-proteins circulate and are the primary source of lipid infoam cells; adipocytes are not known to release lipopro-teins (48,49). Indeed, on the basis of the RNA sequencingand microarray expression studies (Gene Expression Om-nibus database and unpublished data), we have not iden-tified the canonical protein constituents of knownlipoprotein particles (e.g., ApoB) in AT (data not shown).If an endocytic pathway does deliver lipid to lysosomes inATMs, then there must exist a heretofore undefinedmechanism of lipid release by adipocytes.

Our findings were unexpected and point to previouslyunappreciated complexity in the relationship betweenATMs and adipocytes. These studies suggest the existenceof an intratissue lipid cycle not accounted for by currentmodels and point to further areas of study.

Acknowledgments. The authors thank Eleanor Ables (Department ofMedicine, Columbia University) for technical support and Theresa Swayne(Herbert Irving Comprehensive Cancer Center, Columbia University) for assistancewith confocal microscopy and quantification. The authors also thank MasaakiKomatsu (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) forproviding the Atg7 F/F mice. In addition, the authors thank Hong Du (IndianaUniversity, Bloomington, IN) for providing the Lipa2/2 mice.Funding. This research was supported by the National Institutes of Health,National Institute of Diabetes and Digestive and Kidney Diseases, includinggrants R01-DK-066525 and R01-DK-101942, and support from the ColumbiaUniversity Diabetes Research Center (P30-DK-063608), the New York ObesityNutrition Research Center (P30-DK-026687), the Institute of Human Nutrition(T32-DK-007647), and the Irving Institute for Clinical and Translational Research,National Center for Advancing Translational Sciences (UL1-TR-000040).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. A.G. designed the studies, performed all experi-ments other than the lysosomal acid lipase BM experiments, interpreted the results,and wrote the manuscript. X.X. performed lysosomal acid lipase BM experiments.A.W.F. designed the studies, interpreted the results, and wrote the manuscript.A.G. and A.W.F. are the guarantors of this work and, as such, had full accessto all the data in the study and take responsibility for the integrity of the dataand the accuracy of the data analysis.

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