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Rosiglitazone reverses inflammation in epididymal white adipose tissue in hormone-sensitive lipase-knockout mice

Open AccessPublished:October 19, 2022DOI:https://doi.org/10.1016/j.jlr.2022.100305

      Abstract

      Hormone-sensitive lipase (HSL) plays a crucial role in intracellular lipolysis, and loss of HSL leads to diacylglycerol (DAG) accumulation, reduced fatty acid mobilization, and impaired peroxisome proliferator-activated receptor (PPAR) γ signaling. HSL knock-out mice exhibit adipose tissue inflammation, but the underlying mechanisms are still not clear. Here we investigated if and to what extent HSL loss contributes to endoplasmic reticulum (ER) stress and adipose tissue inflammation in HSL knock-out mice. Further, we were interested in how impaired PPARγ signaling affects the development of inflammation in epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) of HSL knock-out mice, and if DAG and ceramide (Cer) accumulation contribute to adipose tissue inflammation and ER stress. Ultrastructural analysis showed a markedly dilated ER in both eWAT and iWAT upon loss of HSL. In addition, HSL knock-out mice exhibited macrophage infiltration and increased F4/80 mRNA expression, a marker of macrophage activation, in eWAT, but not in iWAT. We show treatment with rosiglitazone, a PPARγ agonist, attenuated macrophage infiltration and ameliorated inflammation of eWAT, but expression of ER stress markers remained unchanged, as did DAG and Cer levels in eWAT. Taken together, we show HSL loss promoted ER stress in both eWAT and iWAT of HSL knock-out mice, but inflammation and macrophage infiltration occurred mainly in eWAT. Also, PPARγ activation reversed inflammation, but not ER stress and DAG accumulation. These data indicate that neither reduction of DAG levels nor ER stress contribute to the reversal of eWAT inflammation in HSL knock-out mice.

      KEY WORDS

      ABBREVIATIONS:

      AT (adipose tissue), BAT (brown adipose tissue), Ceramide ((Cer)), DAG (diacylglycerol), eWAT (epididymal), Hprt (hypoxanthin-guanin-phosphoribosyltransferase), HSL (hormone-sensitive lipase white adipose tissue), iWAT (inguinal white adipose tissue), PPARγ (peroxisome proliferator-activated receptor gamma), FA (fatty acids)

      INTRODUCTION

      Hormone-sensitive lipase (HSL) catalyzes the release of fatty acids (FA) from adipose tissue depots in times of energy demand. Upon hormonal stimulation, lipolysis is activated and the enzymes adipose triglyceride lipase (ATGL), HSL and monoacylglyerol lipase successively hydrolyze triacylglycerol (
      • Zechner R.
      FAT FLUX: enzymes, regulators, and pathophysiology of intracellular lipolysis.
      ). HSL has the highest affinity for diacylglycerol (DAG) (,
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      • Waeg G.
      • Wagner E.
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      • Zechner R.
      Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.
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      • Haemmerle G.
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      • Zechner R.
      Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.
      )], reduced FA mobilization and impaired peroxisome proliferator-activated receptor (PPAR) γ signaling (
      • Zimmermann R.
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      ). HSL loss also promotes adipocyte hypertrophy in WAT and leads to decreased overall WAT mass, which occurs due to impaired PPARγ signaling in HSL knock-out mice. Upon aging, HSL knock-out mice show a significant decrease in visceral and subcutaneous WAT mass (
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      Decreased expression and function of adipocyte hormone-sensitive lipase in subcutaneous fat cells of obese subjects.
      ). Decreased HSL activity causes DAG accumulation in HSL knock-out mice (
      • Haemmerle G.
      • Zimmermann R.
      • Hayn M.
      • Theussl C.
      • Waeg G.
      • Wagner E.
      • Sattler W.
      • Magin T.M.
      • Wagner E.F.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.
      ) and in human tissue (
      • Albert J.S.
      • Yerges-Armstrong L.M.
      • Horenstein R.B.
      • Pollin T.I.
      • Sreenivasan U.T.
      • Chai S.
      • Blaner W.S.
      • Snitker S.
      • O’Connell J.R.
      • Gong D.-W.
      • Breyer R.J.
      • Ryan A.S.
      • McLenithan J.C.
      • Shuldiner A.R.
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      • Damcott C.M.
      Null Mutation in Hormone-Sensitive Lipase Gene and Risk of Type 2 Diabetes.
      ). DAG and Cer accumulation has been associated with the development of endoplasmic reticulum (ER) stress, cell death, inflammation and insulin resistance (
      • Kumashiro N.
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      Palmitate mediated diacylglycerol accumulation causes endoplasmic reticulum stress, Plin2 degradation, and cell death in H9C2 cardiomyoblasts.
      ,

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      • Kraegen E.W.
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      Increased malonyl-CoA and diacylglycerol content and reduced AMPK activity accompany insulin resistance induced by glucose infusion in muscle and liver of rats.
      ,
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      ,
      • Mottillo E.P.
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      ). However, the association of HSL loss and DAG or Cer accumulation to ER stress development and inflammation in WAT still needs further clarification.
      Although HSL loss has been extensively studied in mice and humans, the effects of HSL loss on adipose tissue dysfunction and inflammation and also to what extent eWAT and iWAT are affected remain unclear. Further, it is not known yet whether DAG accumulation in WAT of HSL knock-out mice induces ER stress and if it is a cause or consequence of adipose tissue inflammation.
      The aim of this study was to investigate, if and to what extent HSL loss contributes to ER stress and white adipose tissue inflammation in eWAT and iWAT of HSL knock-out mice. Further, we aimed to investigate how impaired PPARγ signaling, affects the development of inflammation in those adipose tissue depots. Additionally we wanted to study to what extent DAG and Cer accumulation contributes to adipose tissue inflammation and ER stress.
      We analyzed ER stress, PPARγ signaling and inflammation in eWAT and iWAT of adult HSL knock-out mice under basal conditions and after chronic rosiglitazone treatment. DAG and Cer levels in eWAT were also analyzed to elucidate the contribution of DAG and Cer accumulation to adipose tissue inflammation and potential ER stress development.

      MATERIAL AND METHODS

      Animals

      HSL knock-out mice were generated by targeted homologous recombination as described previously (
      • Haemmerle G.
      • Zimmermann R.
      • Hayn M.
      • Theussl C.
      • Waeg G.
      • Wagner E.
      • Sattler W.
      • Magin T.M.
      • Wagner E.F.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.
      ) and backcrossed at least 10 times to a C57Bl/6J genetic background. Mice were kept on a standard laboratory chow diet containing 4.5% (w/w) fat (sniff Spezialdiäten GmbH, Soest, Germany). For all studies, male mice with an age range of 10-14 weeks were used. Animals had ad libitum access to food and water and were housed with a light and dark period of 10 and 14 h, respectively, at a room temperature (RT) of 22°C ± 1°C. For re-feeding conditions, mice were fasted for 12 h and had then free access to food for 2 h. Body mass composition was assessed in non-anesthetized mice using the time-domain NMR minispec (Live Mice Analyzer system, Model LF90II, Bruker Optik, Germany). Animals were anesthetized with ISOflo®/isoflurane (Abbott, Abbott Park, IL, USA) and sacrificed by cervical dislocation. All experiments were approved by the Austrian Federal Ministry for Science and the local ethics committee (BMWF-66.007/0017-II/3b/2013).

      Rosiglitazone diet

      For PPARγ agonism, animals were fed standard chow diet containing 200 mg rosiglitazone (Caymen Chemical, USA) per kg diet for 20 days or 28 days. During dietary intervention body weight was monitored regularly.

      Blood parameters

      Blood was collected by retro orbital puncture of anesthetized mice. Plasma levels of non-esterified FA, glycerol and triacylglycerol were measured using the commercially available kits NEFAC (WAKO Chemicals, Germany), TG Infinity Reagent (Thermo Fisher Scientific, USA) and Free Glycerol Reagent (Sigma Aldrich, USA). Plasma leptin was determined using a mouse leptin ELISA (#90030, Chrystal Chem, USA).

      Histological analysis

      eWAT and iWAT tissues were fixed in 10% neutrally buffered formalin solution and tissue was processed in a Tissue-Tek VIP (Sakura, Germany). Three 3 μm thick sections were prepared and attached to charged glass slides (Menzel Superfrost Plus, Thermo Fisher Scientific, USA). Antigen retrieval was performed for 20 min at 90°C in a Decloaking Chamber (DC2012, Biocare Medical, USA) in 10 mM sodium citrate buffer (pH 6) with 0.5% Tween20. Immunohistochemistry was performed with a monoclonal anti-Mac-2 primary antibody (1:500, CL8942AP, Cedarlane, Canada), the Vectastain ABC HRP rat kit (PK-4004, Vector Labs, USA), Sigmafast DAB for visualization (D4293, Sigma-Aldrich, USA) and hematoxylin for counterstaining. Images were captured with an Aperio ScanScope AT (Leica Biosystems, Austria) digital slide scanner at 40-fold magnification. The Slide J plugin (
      • Della Mea V.
      • Baroni G.L.
      • Pilutti D.
      • Di Loreto C.
      SlideJ: An ImageJ plugin for automated processing of whole slide images.
      ) was used in connection with Image J (Fiji distribution) to split the Aperio digital slides into TIFF images (
      • Schindelin J.
      • Arganda-Carreras I.
      • Frise E.
      • Kaynig V.
      • Longair M.
      • Pietzsch T.
      • Preibisch S.
      • Rueden C.
      • Saalfeld S.
      • Schmid B.
      • Tinevez J.Y.
      • White D.J.
      • Hartenstein V.
      • Eliceiri K.
      • Tomancak P.
      • Cardona A.
      Fiji: An open-source platform for biological-image analysis.
      ). The Adiposoft plugin was used to detect and count adipocytes (
      • Galarraga M.
      • Campión J.
      • Munõz-Barrutia A.
      • Boqué N.
      • Moreno H.
      • Martínez J.A.
      • Milagro F.
      • Ortiz-de-Solórzano C.
      Adiposoft: Automated software for the analysis of white adipose tissue cellularity in histological sections.
      ). Crown-like structures (CLS) were counted by two persons on two different sections per sample. CLS were defined by a minimum of three macrophages surrounding an individual adipocyte remnant.

      Transmission electron microscopy

      Small fragments of eWAT and iWAT tissue from perfused mice were fixed in 2% glutaraldehyde-2% paraformaldehyde in phosphate buffer for 4 h at RT, postfixed in 1% osmium tetroxide, dehydrated in a graded series of acetone and embedded in an Epon-Araldite mixture. To determine the region of interest, semithin sections were cut and stained with toluidine blue. Thin sections were obtained with an MT-X Ultratome (RMC; Tucson, AZ, USA), stained with lead citrate, and examined with a CM10 transmission electron microscope (Philips; Eindhoven, The Netherlands).

      Tissue homogenization and western blotting analysis

      Snap frozen tissues were homogenized in ice-cold homogenization buffer (0.25 M sucrose, 1 mM EDTA, 1 mM DTT, pH 7) containing HALT protease and phosphatase inhibitors (Thermo Fisher Scientific, USA) using a hand held disperser (Ultra-Turrax, IKA, Germany). Samples were centrifuged for 15 min at 1,000 x g at 4°C. The infranatant was collected and protein concentration was assayed using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories GmbH, Austria). Proteins were separated using SDS-PAGE and proteins were transferred to a PVDF membrane (Bio-Rad Laboratories GmbH, Austria). Specific proteins were detected using the anti-rabbit peIF2 alpha (Ser51) (1:1000, # 3398, Cell Signaling Technology, Boston, USA) and eIF2 alpha (1:1000, # 5324, Cell Signaling Technology, Boston, USA). Alpha tubulin (1:1000, ab52866 Abcam, UK) was used as a loading control. As secondary antibody HRP-conjugated goat anti-rabbit antibody (Cell Signaling Technology, Boston, USA) was used. Proteins were visualized using Clarity™ substrate and the ChemiDoc™system (Bio-Rad Laboratories GmbH, Austria). Signal density was determined with ImageJ (
      • Schneider C.A.
      • Rasband W.S.
      • Eliceiri K.W.
      NIH Image to ImageJ: 25 years of image analysis.
      ) or directly the ChemiDoc™system (Bio-Rad Laboratories GmbH, Austria).

      RNA isolation and quantitative Real-Time PCR

      Tissue homogenization and RNA isolation of frozen tissues was performed using Qiazol reagent and the RNeasy Mini Kit (both Qiagen Vertriebs GmbH, Austria) according to standard protocols. For gene expression analyses, RNA samples were treated with DNase and reversely transcribed into single stranded cDNA with the iScript™gDNA clear cDNA synthesis kit (Bio-Rad Laboratories GmbH, Austria). cDNA samples were amplified using the SsoAdvanced™ universal SYBR green supermix (Bio-Rad Laboratories GmbH, Austria) or the TaqMan™ Gene Expression Master Mix (Thermo Fisher Scientific, USA) and target gene-specific primer pairs (Sigma Aldrich, Austria) or TaqMan™ probes. Primer sequences are available on request. Real-time PCR was run on the C1000 Thermocycler, using the CFX384 Real Time System (Bio-Rad, Austria). Relative target gene expression was normalized to the ribosomal gene 36b4 or hypoxanthin-guanin-phosphoribosyltransferase (Hprt) and calculated using the method published by Pfaffl (
      • Pfaffl M.W.
      A new mathematical model for relative quantification in real-time RT–PCR.
      ).

      Tissue lipid analysis

      Lipids of weighed tissue explants were extracted twice with 4 ml chloroform/methanol (2/1, v/v) containing 500 pmol butylated hydroxytoluene, 1% acetic acid, and 150 pmol of internal standards (ISTD, 14:0-14:0 DAG, d18:1/17:0 Cer, Avanti Polar Lipids, USA) per sample according to Folch et al. (
      • Folch J.
      • Lees M.
      • Sloane Stanley G.H.
      A simple method for the isolation and purification of total lipides from animal tissues.
      ). Extraction was performed with continuous shaking for 30 min at RT. After addition of 800 μl H2O and further incubation for 30 min at RT, samples were centrifuged at 1,000 x g for 15 min at RT to establish phase separation. The lower organic phase was collected, 2.5 ml chloroform were added to the remaining aqueous phase and a second extraction was performed as described above (30 min at RT with subsequent centrifugation). Combined organic phases were dried under a stream of nitrogen and resolved in 800 μl of methanol/2-propanol/water (6/3/1, v/v/v) for UPLC-MS analysis.
      Chromatographic separation was modified according to Knittelfelder et al. using an ACQUITY-UPLC system (Waters Corporation, UK) equipped with a Kinetex C18 column (2.1x50 mm, 1.7μm; Phenomenex) starting a 15 min linear gradient with 80% solvent A (MeOH/H2O, 1/1, v/v; 10 mM ammonium acetate, 0.1% formic acid, 8 μM phosphoric acid) (
      • Knittelfelder O.L.
      • Weberhofer B.P.
      • Eichmann T.O.
      • Kohlwein S.D.
      • Rechberger G.N.
      A versatile ultra-high performance LC-MS method for lipid profiling.
      ).
      An EVOQ Elite™ triple quadrupole mass spectrometer (Bruker, USA) equipped with an Electrospray ionization source was used for detection. DAG and Cer species were analyzed by selected reaction monitoring using (M+NH4)+ to (RCOO+58)+ of the respective esterified FA as transition (15 eV collision energy, 60 ms, 0.7 resolution for Q1/Q3) for DAG and (M+H)+ to m/z 264 (22 eV collision energy, 60 ms, 0.7 resolution for Q1/Q3) for Cer. Data acquisition was done by MS Workstation (Bruker, Germany). Data were normalized for recovery, extraction- and ionization efficacy by calculating analyte/ISTD ratios (AU) and expressed as AU/g tissue.

      Statistical analysis

      Data are presented as mean + SD. Data were tested for normality and statistical significance was determined using unpaired two-tailed Student’s t-test or two-way ANOVA for multiple comparisons. Multiple testing was corrected by Tukey or Holm-Sidak post-hoc test. Graphs and statistics were prepared in Graph Pad Prism version 8.0 (GraphPad Software, USA). Group differences were considered significant for (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

      RESULTS

      HSL loss promotes ER stress in eWAT and iWAT

      To investigate the contribution of HSL loss to the development of ER stress in adipocytes and adipose tissue, the expression of the ER stress markers Bip/Grp78, Chop, the XBP-1 target genes Erdj4 and Edem and phosphorylation of eIF2 alpha were analyzed in eWAT and iWAT of wild-type and HSL knock-out mice.
      HSL knock-out mice showed a robust increase in ER stress markers in eWAT and iWAT (Figure 1). XBP-1 target gene expression of Erdj4 and Edem in eWAT were significantly increased in HSL knock-out mice compared to wild-type mice (Figure 1 A). Consistent with this finding, Bip/Grp78 and Chop mRNA levels were elevated in eWAT of HSL knock-out mice (Figure 1 A). eIF2alpha phosphorylation was significantly increased (1.9-fold) relative to wild-type mice (Figure 1 B and C). Gene expression of the ER stress markers Erdj4, Bip/Grp78 and Chop were also significantly increased in iWAT, whereas Edem expression only showed a trend towards increased expression (Figure 1 D). In iWAT eIF2alpha phosphorylation was 2-fold increased, similar to the results obtained in eWAT (Figure 1E and 1F).
      Figure thumbnail gr1
      Figure 1ER stress markers are significantly increased in eWAT and iWAT of HSL knock-out mice. A) mRNA expression of Xbp1 target genes Erdj4 and Edem, Bip/Grp78 and Chop in eWAT. Target gene abundance was normalized to 36b4 and Hprt and expressed relative to wild-type of each marker (n = 7-8). B) Representative immunoblot of phosphorylated eIF2alpha (peIF2a) and total eIF2alpha (teIF2a) of eWAT of HSL knock-out mice and wild-type mice. C) Quantification of pEIF2alpha/total peIF2 alpha ratio (n = 8). D) mRNA expression of Xbp1 target genes Erdj4 and Edem, Bip/Grp78 and Chop in iWAT. Target gene abundance was normalized to Hprt and expressed relative to wild-type of each marker (n = 6-8). E) Representative immunoblot of phosphorylated eIF2alpha (peIF2a) and total eIF2alpha (teIF2a) of iWAT of wild-type and HSL knock-out mice. F) Quantification of pEIF2alpha/total peIF2 alpha ratio (n = 8). Data are represented as mean ± SD. Statistical significance was determined using unpaired two-tailed Student’s t test corrected for multiple comparisons using the Holm-Sidak method (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.
      Ultrastructural analyses of eWAT and iWAT confirmed the presence of dilated ER in the cytoplasm of numerous HSL knock-out adipocytes (Figure 2 and Figure 3). A visually evident dilated ER was found in about 50% of unilocular adipocytes in both depots. Moreover, unilocular adipocytes from HSL knock out mice occasionally showed other features of stress, including the presence of cholesterol crystals in their cytoplasm and collagen accumulation close to the plasma membrane. Overall, molecular and morphological data demonstrated that HSL loss led to ER stress in both investigated WAT depots.
      Figure thumbnail gr2
      Figure 2Transmission electron microscopy of eWAT from wild-type and HSL knock-out mice. White adipocytes from wild-type mice (A and B) exhibit normal ER structures, whereas adipocytes from HSL knock-out mice (C and D) contain dilated ER structures. B and D are enlargements of the corresponding areas framed in A and C, respectively.
      Figure thumbnail gr3
      Figure 3Transmission electron microscopy of iWAT from wild-type and HSL knock-out mice. In wild-type mice (A and B), white adipocytes contain normal RER structures. In HSL knock-out mice (C and D), adipocytes exhibit dilated RER structures and signs of cellular stress, including redundant basal lamina and cholesterol crystals.

      HSL loss promotes inflammation in eWAT and reduced PPARγ signaling in eWAT and iWAT

      We analyzed macrophage infiltration, inflammatory marker gene expression and PPARγ expression in eWAT and iWAT of wild-type and HSL knock-out mice in order to study whether ER stress is linked to adipose tissue inflammation and dysfunction.
      eWAT of HSL knock-out mice showed adipocyte hypertrophy and an increased number of CLS, and increased expression of the macrophage marker F4/80 and Itgax/CD11c (Figure 4 A, B, C). Expression of inflammatory factors such as interleukin 6 (IL6) and IL10 was also significantly increased, whereas monocyte-chemoattractant protein 1 (Mcp1) and inducible nitric oxide synthase (iNOS) remained unchanged (Figure 4 C). Although iWAT also showed upregulation of ER stress markers, no signs of tissue inflammation were observed and CLS count and inflammatory marker gene expression was comparable among genotypes (Figure 4 D, E, F). Adipose tissue function was decreased in eWAT of HSL knock-out mice, reflected by decreased PPARγ expression, but unchanged Glut4 expression (Figure 5 A). In iWAT PPARγ and Glut4 expression were slightly but not significantly decreased (Figure 5 B). Despite pronounced eWAT inflammation and dysfunction, overall glucose tolerance and insulin sensitivity were not altered in HSL knock-out mice (Supplementary Figure 1 A, B). However, protein kinase B (AKT) phosphorylation in saline injected HSL knock-out animals was slightly but not significantly decreased in both eWAT and iWAT (Supplementary Figure 1 C, D). Upon insulin stimulation, eWAT of HSL knock-out animals was less responsive and showed less AKT phosphorylation than eWAT from wild-type controls. In iWAT of HSL knock-out mice there was only a slight but not significant reduction of AKT phosphorylation detectable after insulin injection.
      Figure thumbnail gr4
      Figure 4CLS density and inflammatory marker gene expression in eWAT and iWAT of wild-type and HSL knock-out mice. A) Quantification of CLS and B) representative pictures of anti-Mac2 staining of eWAT (n = 4). C) mRNA expression of F4/80, IL10, Mcp1, Itgax/Cd11c, IL6 and iNOS in eWAT of HSL knock-out and wild-type mice (n = 4-8). D) Quantification of CLS (n = 3) and E) representative pictures of anti-Mac2 staining of iWAT. F) mRNA expression of F4/80, IL10, Mcp1, Itgax/Cd11c, IL6 and iNOS in iWAT of HSL knock-out and wild-type mice (n = 4-8).PPARγ. The mRNA expression of inflammatory markers was measured by quantitative real-time PCR. Target gene abundance was normalized to 36b4 and/or Hprt and expressed relative to wild-type levels of each marker. Data are represented as mean ± SD. Statistical significance was determined using unpaired two-tailed Student’s t test, corrected for multiple comparisons using the Holm-Sidak method (*) p < 0.05; (**) p < 0.01.
      Figure thumbnail gr5
      Figure 5PPARγ und Glut4 gene expression in eWAT and iWAT of wild-type and HSL knock-out mice. A) mRNA expression of PPARγ and Glut4 in eWAT of HSL knock-out and wild-type mice (n = 7-8). B) mRNA expression of PPARγ and Glut4 in iWAT of HSL knock-out and wild-type mice (n = 4-8). The mRNA expression was measured by quantitative real-time PCR. Target gene abundance was normalized to 36b4 and/or Hprt and expressed relative to wild-type levels of each marker. Data are represented as mean ± SD. Statistical significance was determined using unpaired two-tailed Student’s t test (***) p < 0.001.

      PPARγ activation attenuates inflammation but not ER stress in eWAT

      We hypothesized that decreased PPARγ signaling in eWAT and iWAT might be causal for ER stress and inflammation. To investigate whether ER stress and inflammation are ameliorated in eWAT and iWAT we fed wild-type mice and HSL knock-out a rosiglitazone-enriched diet. eWAT and iWAT of the rosiglitazone-treated wild-type and HSL knock-out mice were analyzed in terms of ER stress marker distribution and adipose tissue inflammation, and compared to untreated wild-type and HSL knock-out mice.
      Rosiglitazone treatment did not affect body weight in wild-type and HSL knock-out mice. eWAT mass was slightly decreased in untreated wild-type mice compared to untreated HSL knock-out littermates (Figure 6 A and B). Mac-2 stainings and CLS quantification of eWAT sections showed that 20 days of rosiglitazone treatment diminished macrophage infiltration and CLS formation in eWAT of HSL knock-out mice (Figure 6 C and D) but still remained higher in HSL knock-out mice. In line with this, F4/80 and IL10 expression was significantly decreased after rosiglitazone treatment (Figure 6 E). Despite decreased inflammation, ER stress marker expression remained at similar levels when compared to wild-type mice (Figure 6 F). PPARγ expression, however, was not affected (Figure 6 G). In iWAT of rosiglitazone treated HSL knock-out mice, rosiglitazone treatment had no effect on iWAT mass or inflammatory marker expression (Supplementary Figure 2 A, B), but ER stress markers seemed to be even slightly increased upon rosiglitazone treatment (Supplementary Figure 2 C). PPARγ expression was significantly increased in wild-type mice upon rosiglitazone treatment but only showed a slight increase in HSL knock-out mice (Supplementary Figure 2 D). Gene expression of the PPARγ targets leptin, Plin1 and adiponectin (AdipoQ) were decreased in HSL knock-mice compared to wild-type controls and showed a trend towards up-regulation upon rosiglitazone treatment in eWAT and iWAT except for leptin (Supplementary Figure 3). In eWAT Plin1 was significantly up-regulated in HSL knock-out mice upon rosiglitazone treatment (Supplementary Figure 3).
      Figure thumbnail gr6
      Figure 6Body weight, inflammatory marker, ER stress marker and PPARγ expression in eWAT after rosiglitazone treatment of HSL knock-out mice. A) Body weight and B) eWAT wet weight of wild-type and HSL knock-out mice without (- Rosi) and with rosiglitazone treatment (+ Rosi) (n = 6-9). C) CLS count in eWAT of wild-type and HSL knock-out mice without (- Rosi) and with rosiglitazone treatment (+ Rosi) (n = 2-5) and D) representative pictures of anti-Mac2 staining of eWAT of wild-type and HSL knock-out mice without (- Rosi) and with rosiglitazone treatment (+ Rosi). E) mRNA expression of inflammatory markers (F4/80, IL10) of wild-type and HSL knock-out mice without (- Rosi) and with rosiglitazone treatment (+ Rosi). F) ER stress marker and G) PPARγ mRNA expression of wild-type and HSL knock-out mice without (- Rosi) and with rosiglitazone treatment (+ Rosi) (n = 6-9). The mRNA expression was measured by quantitative real-time PCR. Target gene abundance was normalized to 36b4 and Hprt and expressed relative to wild-type levels of each marker. Data are given as mean ± SD. Statistical significance was determined using two-way ANOVA and Tukey’s multiple comparisons test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

      Rosiglitazone treatment decreased post-prandial increase in plasma FA and normalized leptin levels but did not affect DAG or Cer accumulation in eWAT of HSL knock-out mice

      Plasma FA were significantly elevated in re-fed HSL knock-out mice. This effect was completely missing in HSL knock-out mice treated with rosiglitazone (Figure 7 A). No significant changes in plasma glycerol (Figure 7 B) and plasma TAG (Figure 7 C) were detected. In ad libitum fed HSL knock-out mice plasma FA and TAG levels remained unchanged, but were significantly decreased upon rosiglitazone treatment in HSL knock-out mice (Supplementary Figure 4). Leptin levels were significantly decreased in HSL knock-out mice but reached wild-type levels upon rosiglitazone treatment (Figure 7 D), which indicates partially restored adipose tissue function.
      Figure thumbnail gr7
      Figure 7Plasma concentrations of FA, glycerol, triacylglycerol (TAG) and leptin in wild-type and HSL knock-out mice treated without or with rosiglitazone. A) plasma FA, B) plasma glycerol, C) plasma TAG and D) plasma leptin levels of wild-type and HSL knock-out mice after 2 h of re-feeding with (+ Rosi) or without rosiglitazone (- Rosi) (n = 6-10). Data are represented as mean ± SD. Statistical significance was determined by two-way ANOVA and Tukey’s multiple comparisons test (*) p < 0.05; (**) p < 0.01.
      Since inflammation and ER stress were more pronounced in eWAT, we analyzed DAG and Cer levels of eWAT from wild-type and HSL knock-out mice with and without rosiglitazone treatment. We found that DAG accumulation was pronounced in eWAT of HSL knock-out mice. Rosiglitazone treatment did not affect the total DAG levels in eWAT of HSL knock-out mice but slightly increased total DAG in wild-type mice (Figure 8 A). No robust effect of rosiglitazone treatment on FA species distribution in the most abundant DAG species (Figure 8 B) in HSL knock-out mice was detected. Overall, significant differences in the DAG FA species distribution occurred among genotypes. DAG 32:0 (16:0/16:0) and DAG 34:1 (16:0/18:1) levels were significantly decreased in HSL knock-out compared to wild-type mice, whereas DAG 36:2 (18:1/18:1) and DAG 36:4 (18:2/18:2) levels were significantly increased (Figure 8 B). The differences among genotypes largely remained unaffected by rosiglitazone treatment. Only DAG 36:4 (18:2/18:2) levels were increased in eWAT of HSL knock-out mice and reduced to wild-type mice levels after rosiglitazone treatment (Figure 8 B). DAG 34:1 (16:0/18:1) levels were significantly decreased in HSL knock-out mice but slightly increased with rosiglitazone treatment, whereas wild-type levels remained unaffected by the treatment. Total eWAT Cer levels did not differ with respect to genotypes and treatment (Figure 8 C). Similar to DAG FA species distribution, also Cer FA species distribution either showed only a treatment effect, where rosiglitazone treatment only altered Cer levels without any difference among genotypes. Cer 34:1 (d18:1/16:0) and Cer 40:1 (d18:1/22:0) levels were slightly decreased by rosiglitazone treatment and Cer 36:1 (d18:1/18:0) levels were increased but almost no changes among genotypes occurred. Cer 42:1 (d18:1/24:0) levels were significantly decreased in HSL knock-out mice, but not affected by rosiglitazone treatment. Cer 42:2 (d18:1/24:1) levels were increased in untreated HSL knock-out mice and only showed a slight trend of higher levels after rosiglitazone treatment (Figure 8 D).
      Figure thumbnail gr8
      Figure 8DAG and Cer levels in eWAT of untreated and rosiglitazone treated wild-type and HSL knock-out mice. A) Total DAG levels of eWAT of wild-type and HSL knock-out mice without (- Rosi) and with rosiglitazone treatment (+ Rosi) and B) DAG species distribution given as % of total DAG signal (n = 4-5). C) Total Cer levels of eWAT of wild-type and HSL knock-out mice without (- Rosi) and with rosiglitazone treatment (+ Rosi), and D) Cer species distribution given as % of total Cer signal (n = 4-5). Data are represented as Mean ± SD. Statistical significance was determined by two-way ANOVA and Tukey’s multiple comparisons test (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

      DISCUSSION

      Data from the present study showed that ER stress occurred to the same extent in both eWAT and iWAT of HSL knock-out mice. We observed macrophage infiltration and increased inflammatory marker expression in eWAT, where rosiglitazone treatment attenuated inflammation but not ER stress. Rosiglitazone treatment did not affect DAG or Cer deposition in eWAT of HSL knock-out mice.
      The expression of ER stress markers and the ultrastructural analyses of eWAT and iWAT demonstrated pronounced ER stress in both investigated WAT depots of HSL knock-out mice. Previous work from Pinent et al and Mottillo et al has indicated that altered HSL activity might interfere with ER stress response (
      • Pinent M.
      • Hackl H.
      • Burkard T.R.
      • Prokesch A.
      • Papak C.
      • Scheideler M.
      • Hämmerle G.
      • Zechner R.
      • Trajanoski Z.
      • Strauss J.G.
      Differential transcriptional modulation of biological processes in adipocyte triglyceride lipase and hormone-sensitive lipase-deficient mice.
      ,
      • Mottillo E.P.
      • Shen X.J.
      • Granneman J.G.
      beta3-adrenergic receptor induction of adipocyte inflammation requires lipolytic activation of stress kinases p38 and JNK.
      ). They have shown that loss of HSL or ATGL differentially affects transcription of several metabolic genes in various tissues such as WAT, heart or liver tissue (
      • Pinent M.
      • Hackl H.
      • Burkard T.R.
      • Prokesch A.
      • Papak C.
      • Scheideler M.
      • Hämmerle G.
      • Zechner R.
      • Trajanoski Z.
      • Strauss J.G.
      Differential transcriptional modulation of biological processes in adipocyte triglyceride lipase and hormone-sensitive lipase-deficient mice.
      ). Microarray analysis of WAT and further gene ontology annotation has shown that ER stress response was upregulated in WAT of HSL knock-out mice (
      • Pinent M.
      • Hackl H.
      • Burkard T.R.
      • Prokesch A.
      • Papak C.
      • Scheideler M.
      • Hämmerle G.
      • Zechner R.
      • Trajanoski Z.
      • Strauss J.G.
      Differential transcriptional modulation of biological processes in adipocyte triglyceride lipase and hormone-sensitive lipase-deficient mice.
      ), but the underlying mechanisms have not been investigated. Mottillo et al have suggested that inflammatory response and ER stress in adipose tissue in vivo and in adipocytes in vitro is promoted by fully activated lipolysis. The selective HSL inhibitor suppressed activated lipolysis and was in turn suppressing inflammation but not ER stress. HSL inhibition rather tended to further increase ER stress than inhibiting it (
      • Mottillo E.P.
      • Shen X.J.
      • Granneman J.G.
      beta3-adrenergic receptor induction of adipocyte inflammation requires lipolytic activation of stress kinases p38 and JNK.
      ). In contrast to Mottillo et al, chronic loss of HSL activity as it occurs in HSL knock-out mice did not prevent but induced inflammation in WAT (
      • Cinti S.
      • Mitchell G.
      • Barbatelli G.
      • Murano I.
      • Ceresi E.
      • Faloia E.
      • Wang S.
      • Fortier M.
      • Greenberg A.S.
      • Obin M.S.
      Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.
      ,
      • Hansson O.
      • Ström K.
      • Güner N.
      • Wierup N.
      • Sundler F.
      • Höglund P.
      • Holm C.
      • Stro K.
      • Gu N.
      • Wierup N.
      • Sundler F.
      • Holm C.
      Inflammatory Response in White Adipose Tissue in the Non-Obese Hormone-Sensitive Lipase Null Mouse Model research articles.
      ).
      Our and previous work showed that HSL knock-out mice exhibit adipose tissue inflammation (
      • Cinti S.
      • Mitchell G.
      • Barbatelli G.
      • Murano I.
      • Ceresi E.
      • Faloia E.
      • Wang S.
      • Fortier M.
      • Greenberg A.S.
      • Obin M.S.
      Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.
      ,
      • Hansson O.
      • Ström K.
      • Güner N.
      • Wierup N.
      • Sundler F.
      • Höglund P.
      • Holm C.
      • Stro K.
      • Gu N.
      • Wierup N.
      • Sundler F.
      • Holm C.
      Inflammatory Response in White Adipose Tissue in the Non-Obese Hormone-Sensitive Lipase Null Mouse Model research articles.
      ). However, we found that inflammation mainly occured in the visceral adipose tissue depot eWAT and did not occur in iWAT under the present conditions. Previous data from our group also indicated that eWAT inflammation is not associated to systemic inflammation, since inflammatory cytokines were not increased in plasma of HSL knock-out mice (Supplementary Figure 5). Previous studies from Cinti et al and Hansson et al have also observed inflammation in visceral adipose tissue (
      • Cinti S.
      • Mitchell G.
      • Barbatelli G.
      • Murano I.
      • Ceresi E.
      • Faloia E.
      • Wang S.
      • Fortier M.
      • Greenberg A.S.
      • Obin M.S.
      Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans.
      ,
      • Hansson O.
      • Ström K.
      • Güner N.
      • Wierup N.
      • Sundler F.
      • Höglund P.
      • Holm C.
      • Stro K.
      • Gu N.
      • Wierup N.
      • Sundler F.
      • Holm C.
      Inflammatory Response in White Adipose Tissue in the Non-Obese Hormone-Sensitive Lipase Null Mouse Model research articles.
      ), but we also investigated subcutaneous adipose tissue. The data from our study revealed that ER stress occurred to the same extent in eWAT and iWAT, but macrophage infiltration and inflammation were only present in eWAT. This supports the concept that pronounced ER stress does not cause initiation of adipose tissue inflammation, at least in WAT. A previous study from our group showed that ATGL knock-out mice have massive inflammation in brown adipose tissue without any signs of ER stress, indicating that adipose tissue inflammation and ER stress are not consequently linked (
      • Kotzbeck P.
      • Giordano A.
      • Mondini E.
      • Murano I.
      • Severi I.
      • Venema W.
      • Cecchini M.P.
      • Kershaw E.E.
      • Barbatelli G.
      • Haemmerle G.
      • Zechner R.
      • Cinti S.
      Brown adipose tissue whitening leads to brown adipocyte death and adipose tissue inflammation.
      ).
      PPARγ and respective target gene expression was reduced in eWAT and iWAT indicating reduced adipose tissue functionality in both WAT depots. Treatment of HSL knock-out mice with the PPARγ agonist rosiglitazone diminished inflammation in eWAT of HSL knock-out mice, but did not resolve ER stress. Rosiglitazone treatment also partially restored expression of PPARγ target genes in eWAT and iWAT (Supplementary Figure 5) and normalized plasma leptin levels. Shen et al have shown that FAs cleaved by HSL are needed for PPARγ activation (
      • Shen W.J.
      • Yu Z.
      • Patel S.
      • Jue D.
      • Liu L.F.
      • Kraemer F.B.
      Hormone-sensitive lipase modulates adipose metabolism through PPARγ.
      ). In line with these observations, we found lowered eWAT mass in HSL knock-out mice, which was partially restored upon rosiglitazone treatment. Interestingly, our data indicate that adipocyte loss and inflammation predominantly occurs in eWAT, where PPARγ signaling seemed to be decreased to a greater extent in eWAT than in iWAT. In high-fat diet fed adipocyte-specific HSL knock-out mice however, eWAT and iWAT inflammation occurred at the same time, but it seems that inflammation was much more pronounced in eWAT while PPARγ signaling was decreased in both depots (
      • Pajed L.
      • Taschler U.
      • Tilp A.
      • Hofer P.
      • Kotzbeck P.
      • Kolleritsch S.
      • Radner F.P.W.
      • Pototschnig I.
      • Wagner C.
      • Schratter M.
      • Eder S.
      • Huetter S.
      • Schreiber R.
      • Haemmerle G.
      • Eichmann T.O.
      • Schweiger M.
      • Hoefler G.
      • Kershaw E.E.
      • Lass A.
      • Schoiswohl G.
      Advanced lipodystrophy reverses fatty liver in mice lacking adipocyte hormone-sensitive lipase.
      ). We speculate that upon long term high-fat diet challenge both depots are similarly affected.
      Although we and others found significant DAG accumulation in adipose tissue of HSL knock-out mice, our results did not identify DAG or Cer lipotoxicity as source of inflammation. We found that neither DAG nor Cer levels in eWAT were altered by rosiglitazone treatment. This is in line with results from Shen et al who did not detect any changes in DAG levels upon rosiglitazone treatment of HSL knock-out mice. (
      • Shen W.J.
      • Yu Z.
      • Patel S.
      • Jue D.
      • Liu L.F.
      • Kraemer F.B.
      Hormone-sensitive lipase modulates adipose metabolism through PPARγ.
      ). We found a shifted distribution of DAG species, with DAG 18:2/18:2 levels elevated in untreated HSL knock-out mice which was reversed upon rosiglitazone treatment. There is evidence that DAG and Cer accumulation in general and also accumulation of some DAG species like DAG 18:2/18:2 contribute to the development of insulin resistance in muscle in vivo (
      • Zabielski P.
      • Chacinska M.
      • Charkiewicz K.
      • Baranowski M.
      • Gorski J.
      • Blachnio-Zabielska A.U.
      Effect of metformin on bioactive lipid metabolism in insulin-resistant muscle.
      ). However, if and how DAGs contribute to insulin resistance is still a matter of debate, since other studies have found no association of increased tissue DAG levels with insulin resistance (
      • Anastasiou C.A.
      • Kavouras S.A.
      • Lentzas Y.
      • Gova A.
      • Sidossis L.S.
      • Melidonis A.
      Diabetes mellitus is associated with increased intramyocellular triglyceride, but not diglyceride, content in obese humans.
      ). Increased DAG levels in macrophages have even suppressed an inflammatory burst and might be important in preventing sepsis in vivo (
      • Wang Y.F.
      • Lee G.L.
      • Huang Y.H.
      • Kuo C.C.
      sn-1,2-diacylglycerols protect against lethal endotoxemia by controlling systemic inflammation.
      ). DAG and Cer concentrations are not or only partially responsible for ER stress and adipose tissue inflammation in HSL knock-out mice according to our data. However, since rosiglitazone treatment did not ameliorate ER stress and did not reduce DAG accumulation we cannot exclude that DAG accumulation might drive ER stress. Previous studies have shown that ER stress is driven by DAG accumulation in vitro in cardiomyocytes (
      • Akoumi A.
      • Haffar T.
      • Mousterji M.
      • Kiss R.S.
      • Bousette N.
      Palmitate mediated diacylglycerol accumulation causes endoplasmic reticulum stress, Plin2 degradation, and cell death in H9C2 cardiomyoblasts.
      ). In line with our findings, pioglitazone treatment in humans, did not affect overall Cer levels in adipose tissue. Although sphingosine (d16:0) and (d18:1) containing ceramides have been previously described for both eWAT and iWAT (
      • Muralidharan S.
      • Shimobayashi M.
      • Ji S.
      • Burla B.
      • Hall M.N.
      • Wenk M.R.
      • Torta F.
      A reference map of sphingolipids in murine tissues.
      ), we primarily focused on sphingosine (d18:1) containing ceramides. Multiple studies were able to show, that especially Cer d18:1 species, were associated to insulin resistance in humans (
      • Bergman B.C.
      • Brozinick J.T.
      • Strauss A.
      • Bacon S.
      • Kerege A.
      • Bui H.H.
      • Sanders P.
      • Siddall P.
      • Wei T.
      • Thomas M.K.
      • Kuo M.S.
      • Perreault L.
      Muscle sphingolipids during rest and exercise: a C18:0 signature for insulin resistance in humans.
      ,
      • Tonks K.T.
      • Coster A.C.
      • Christopher M.J.
      • Chaudhuri R.
      • Xu A.
      • Gagnon-Bartsch J.
      • Chisholm D.J.
      • James D.E.
      • Meikle P.J.
      • Greenfield J.R.
      • Samocha-Bonet D.
      Skeletal muscle and plasma lipidomic signatures of insulin resistance and overweight/obesity in humans.
      ,
      • Hilvo M.
      • Salonurmi T.
      • Havulinna A.S.
      • Kauhanen D.
      • Pedersen E.R.
      • Tell G.S.
      • Meyer K.
      • Teeriniemi A.M.
      • Laatikainen T.
      • Jousilahti P.
      • Savolainen M.J.
      • Nygård O.
      • Salomaa V.
      • Laaksonen R.
      Ceramide stearic to palmitic acid ratio predicts incident diabetes.
      ) and Cer d18:1/16:0 and Cer d18:1/18:0 were specifically more abundant in eWAT of high fat diet fed mice (
      • Muralidharan S.
      • Shimobayashi M.
      • Ji S.
      • Burla B.
      • Hall M.N.
      • Wenk M.R.
      • Torta F.
      A reference map of sphingolipids in murine tissues.
      ).We found that rosiglitazone treatment decreased postprandial plasma FA concentration in HSL knock-out mice, which could be associated with reduction of adipose tissue inflammation. A previous study from Zimmermann et al has shown that decreased PPARγ signaling impaired FA re-esterification in WAT and thereby compensated the lipolytic defect in HSL knock-out mice (
      • Zimmermann R.
      • Haemmerle G.
      • Wagner E.M.
      • Strauss J.G.
      • Kratky D.
      • Zechner R.
      Decreased fatty acid esterification compensates for the reduced lipolytic activity in hormone-sensitive lipase-deficient white adipose tissue.
      ). Moreover, they have reported elevated palmitate levels in adipose tissue (
      • Haemmerle G.
      • Zimmermann R.
      • Hayn M.
      • Theussl C.
      • Waeg G.
      • Wagner E.
      • Sattler W.
      • Magin T.M.
      • Wagner E.F.
      • Zechner R.
      Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.
      ). Increased palmitate levels affected by lowered PPARγ signaling could contribute to adipose tissue inflammation by inducing inflammatory signaling (
      • Ajuwon K.M.
      • Spurlock M.E.
      Palmitate activates the NF-kappaB transcription factor and induces IL-6 and TNFalpha expression in 3T3-L1 adipocytes.
      ). Although an overload in FAs, especially palmitate, has been associated with the development of ER stress (
      • Guo W.
      • Wong S.
      • Xie W.
      • Lei T.
      • Luo Z.
      Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat primary preadipocytes.
      ), decreased postprandial plasma FA concentration in HSL knock-out mice did not affect ER stress markers in eWAT and iWAT. Recently, Chitraju et al have demonstrated that diacylglycerol-O-acyltransferase-1 (DGAT-1)-mediated FA re-esterification is crucial to prevent FA induced ER stress during maximal lipolysis in adipocytes (
      • Chitraju C.
      • Mejhert N.
      • Haas J.T.
      • Diaz-Ramirez L.G.
      • Grueter C.A.
      • Imbriglio J.E.
      • Pinto S.
      • Koliwad S.K.
      • Walther T.C.
      • V Farese R.
      Triglyceride Synthesis by DGAT1 Protects Adipocytes from Lipid-Induced ER Stress during Lipolysis.
      ). Loss of DGAT-1, specifically in adipocytes, induced ER stress and inflammation in WAT by suppressing re-esterification and FA detoxification. ER stress was not suppressed in our study, although we found lowered postprandial plasma FA concentrations, and therefore most likely efficient re-esterification in rosiglitazone-treated mice. Since our study was carried out under postprandial conditions when lipolysis is suppressed we speculate that ER stress is induced by different mechanisms during maximal lipolysis or postprandial.
      Lipolysis-derived FAs are crucial for PPARγ activation (
      • Shen W.J.
      • Yu Z.
      • Patel S.
      • Jue D.
      • Liu L.F.
      • Kraemer F.B.
      Hormone-sensitive lipase modulates adipose metabolism through PPARγ.
      ); a shift in lipolytic activity in mice and humans might affect adipogenesis and adipose tissue functionality in vivo. A study in humans demonstrated that female obesity alters lipolytic activity in subcutaneous adipose tissue which goes hand in hand with diminished functionality, impaired glucose metabolism and weight gain (
      • Arner P.
      • Andersson D.P.
      • B€ Ackdahl J.
      • Dahlman I.
      • Rydé M.
      • Bäckdahl J.
      • Dahlman I.
      • Rydén M.
      Weight Gain and Impaired Glucose Metabolism in Women Are Predicted by Inefficient Subcutaneous Fat Cell Lipolysis.
      ). Data obtained from an Amish population with various mutants of HSL showed that the HSL loss in humans leads to impaired glucose metabolism (
      • Albert J.S.
      • Yerges-Armstrong L.M.
      • Horenstein R.B.
      • Pollin T.I.
      • Sreenivasan U.T.
      • Chai S.
      • Blaner W.S.
      • Snitker S.
      • O’Connell J.R.
      • Gong D.-W.
      • Breyer R.J.
      • Ryan A.S.
      • McLenithan J.C.
      • Shuldiner A.R.
      • Sztalryd C.
      • Damcott C.M.
      Null Mutation in Hormone-Sensitive Lipase Gene and Risk of Type 2 Diabetes.
      ), a status that is not fully supported by our data in mice. Further, impaired HSL function decreases subcutaneous adipose tissue mass in humans (
      • Albert J.S.
      • Yerges-Armstrong L.M.
      • Horenstein R.B.
      • Pollin T.I.
      • Sreenivasan U.T.
      • Chai S.
      • Blaner W.S.
      • Snitker S.
      • O’Connell J.R.
      • Gong D.-W.
      • Breyer R.J.
      • Ryan A.S.
      • McLenithan J.C.
      • Shuldiner A.R.
      • Sztalryd C.
      • Damcott C.M.
      Null Mutation in Hormone-Sensitive Lipase Gene and Risk of Type 2 Diabetes.
      ), whereas in our study primarily visceral adipose tissue mass declined in mice. But impaired HSL activity has also been associated with inflammation and decreased expression of PPARγ target gene in subcutaneous adipose tissue (
      • Albert J.S.
      • Yerges-Armstrong L.M.
      • Horenstein R.B.
      • Pollin T.I.
      • Sreenivasan U.T.
      • Chai S.
      • Blaner W.S.
      • Snitker S.
      • O’Connell J.R.
      • Gong D.-W.
      • Breyer R.J.
      • Ryan A.S.
      • McLenithan J.C.
      • Shuldiner A.R.
      • Sztalryd C.
      • Damcott C.M.
      Null Mutation in Hormone-Sensitive Lipase Gene and Risk of Type 2 Diabetes.
      ). Data from our studies on HSL knock-out mice and also data from studies on human HSL functionality (
      • Albert J.S.
      • Yerges-Armstrong L.M.
      • Horenstein R.B.
      • Pollin T.I.
      • Sreenivasan U.T.
      • Chai S.
      • Blaner W.S.
      • Snitker S.
      • O’Connell J.R.
      • Gong D.-W.
      • Breyer R.J.
      • Ryan A.S.
      • McLenithan J.C.
      • Shuldiner A.R.
      • Sztalryd C.
      • Damcott C.M.
      Null Mutation in Hormone-Sensitive Lipase Gene and Risk of Type 2 Diabetes.
      ,
      • Arner P.
      • Andersson D.P.
      • B€ Ackdahl J.
      • Dahlman I.
      • Rydé M.
      • Bäckdahl J.
      • Dahlman I.
      • Rydén M.
      Weight Gain and Impaired Glucose Metabolism in Women Are Predicted by Inefficient Subcutaneous Fat Cell Lipolysis.
      ) demonstrate that HSL is indispensable for adipose tissue function and that a shift in HSL activity, for example in obesity, causes impaired adipose tissue function and inflammation, and is possibly responsible for the development of obesity-related disorders. HSL seems to have a certain depot-specificity that appears to be different in mice and humans.

      CONCLUSION

      HSL loss promoted ER stress in both visceral (eWAT) and subcutaneous adipose tissue (iWAT) to the same extent. Inflammation and macrophages infiltration were only detected in eWAT, and PPARγ activation attenuated inflammation but not ER stress in eWAT. Rosiglitazone treatment did not affect the DAG or Cer accumulation in eWAT of HSL knock-out mice, but it altered plasma FA concentration. We assume that reduced FA re-esterification in HSL knock-out mice promotes inflammatory signaling in eWAT, which is reversed by rosiglitazone treatment. Further studies on HSL depot-specificity in mice and humans are necessary to fully understand the role of HSL in obesity and associated comorbidities.

      DATA AVAILABILITY

      The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. All data generated and analyzed during this study are included in this published article (and its supplementary information files).

      Acknowledgements:

      We thank Kathrin A. Zierler, and Birgit Juritsch for animal care and genotyping and Lukas Pein for CLS quantification.

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