Inflammatory Response in White Adipose Tissue in the Non-Obese Hormone-Sensitive Lipase Null Mouse Model Ola Hansson,*,† Kristoffer Stro1 m,† Nuray Gu1 ner,‡ Nils Wierup,† Frank Sundler,† Peter Ho1 glund,‡ and Cecilia Holm† Department of Experimental Medical Science, Lund University, BMC C11, SE-221 84 Lund, Sweden, and Department of Clinical Sciences, Lund University Hospital, Lund, Sweden Received March 17, 2006
In the present study, a local inflammatory response in white adipose tissue from the nonobese HSLnull mouse model is demonstrated. The protein levels of several well-known markers of inflammation, like TNFR and ferritin HC, were highly increased and accompanied by an activation of NFκB. A number of macrophage proteins, i.e., gal-3, Capg, and MCP-4, were expressed at increased levels and immunohistochemical analyses revealed an increased infiltration of F4/80+ cells. Keywords: hormone-sensitive lipase • white adipose tissue • proteomics • retinoic acid • inflammation
Hormone-sensitive lipase is a key enzyme in the mobilization of fatty acids from lipid stores in adipocytes as well as nonadipocytes. Expression of HSL has been demonstrated in various tissues, including white adipose tissue (WAT),1 brown adipose tissue (BAT),2 myocytes,1 pancreatic β-cells,3 and testis,4 with the highest level in WAT. One distinctive feature of HSL among lipases is its broad substrate specificity. Besides acylglycerols,5 HSL has the ability to hydrolyze cholesteryl esters,5 steroid esters,6 and retinyl esters.7 Several HSL-null mouse models have been created in recent years.8-11 Among the described characteristics of these models are male sterility,8-10 diglyceride accumulation in various tissues,9 and resistance to diet-induced obesity.12,13 These features have all been verified in the HSL-null mouse model investigated here, which has furthermore been shown to exhibit moderate impairment of insulin sensitivity in all insulin target tissues investigated, i.e., liver, skeletal muscle, and WAT11 and unpublished results. This impairment was manifested as perturbation of the ability of insulin to suppress hepatic glucose production, reduced insulinstimulated glucose uptake in skeletal muscle and reduction of insulin-stimulated lipogenesis in WAT.11 The link between obesity and insulin resistance is well established and growing evidence also connects inflammation to obesity, insulin resistance, and type 2 diabetes mellitus.14 In WAT of obese individuals increased expression of a number of proinflammatory molecules, including interleukin-6,15 transforming growth factor-beta,16 and tumor necrosis factor-alpha (TNF-R),17 has been reported. However, the source of these molecules has been debated. It has been suggested that they are secreted by adipocytes, by infiltrating inflammatory cells,
e.g., macrophages, or by both. A strong association between obesity and increased number of bone marrow-derived macrophages has been reported.18 In this study, the authors estimated that the percentage of macrophages in adipose tissue ranges from 10% in lean mice to over 50% in extremely obese, leptin-deficient, mice.18 This estimation was based on a global analysis of the transcriptome, showing that a large number of the significantly regulated genes were macrophage and inflammatory genes. However, one critical question is how the inflammatory response is triggered and maintained in WAT in insulin resistant and obese states and what mechanisms could attract macrophages to WAT. It has previously been reported that the mRNA levels of TNFR are increased in WAT from HSL-null mice, suggesting that there is WAT inflammation in this nonobese, insulin-resistant model.12 To characterize the inflammatory state of this mouse model further, we chose to investigate the protein expression profile of WAT using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). From a methodological point of view, analyzing data generated by 2DPAGE involves many steps, including spot detection, spot matching, background subtraction, and normalization. In the process of data analysis, a decision has to be made concerning the combination of methods that are the most suitable to apply to the data set in order to get reliable quantification of proteins. This is an important issue that is often ignored and inadequately documented in 2D-PAGE publications. When analyzing 2D-PAGE data, it is also important to consider how to handle missing data points. Is it a biological or a technical reason for the missing data points? Both these issues have been investigated and are reported in the supplementary section of this paper.
* To whom correspondence should be addressed. Department of Experimental Medical Science, Division of Diabetes, Metabolism and Endocrinology, Lund University, BMC C11, SE 221 84 Lund, Sweden. Tel: +46 46 2229772. Fax: +46 46 2224022. E-mail:
[email protected]. † Department of Experimental Medical Science, Lund University. ‡ Department of Clinical Sciences, Lund University Hospital.
The aim of this study was to investigate the protein expression profile of WAT from HSL-null mice in comparison with wild-type littermates, fed either normal chow diet (ND) or high fat diet (HFD), to further explore the role of HSL in WAT biology and inflammation.
Introduction
10.1021/pr060101h CCC: $33.50
2006 American Chemical Society
Journal of Proteome Research 2006, 5, 1701-1710
1701
Published on Web 05/27/2006
research articles Experimental Section Animals. The animals used in this study had a mixed genetic background from the inbred strains C57BL/6 J and SV129.11 The animals were fed either ND or HFD prior to the time of sacrifice. In the ND, 10.6% of the energy originated from fat and 73.1% from carbohydrate. In the HFD, 58.0% of the energy originated from fat and 25.6% from carbohydrate (Research diets incorporated). The mice had free access to food and water at all times. When sacrificed the animals were anaesthetized using midazolam 0.4 mg/mouse (Dormicum, Hoffman-La-Roche, Basel, Switzerland) in combination with fluanison 0.9 mg/mouse and fentanyl 0.02 mg/mouse (Hypnorm, Janssen, Beerse, Belgium) and killed with cervical dislocation. The studies were approved by the local Animal Ethics Committee. One-Dimensional Gel Electrophoresis. Parametrial WAT from 13-months old female mice, fed a HFD for 40 weeks prior to the time of sacrifice, was homogenized using a PotterElvehjem homogenizer (400 rpm, 10 strokes). The samples were centrifuged (10 000 × g, 4 °C, 30 min) and 100 µg of total protein from the pellet fractions were separated using one-dimensional polyacrylamide gel electrophoresis (1D-PAGE) on a gradient gel (10-20%). The gel was stained according to.19 Briefly, the gel was fixed for 1 h in acetic acid, methanol and H2O (70:500: 430) and then stained for 3 h in 2% phosphoric acid, 10% ammonium sulfate, 0.1% coomassie brilliant blue G-250:MeOH (4:1). After washing in deionized water, bands were excised and identified by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF-MS). Two-Dimensional Gel Electrophoresis. Epididymal fat pads from four 12-14 months old male mice, fed either a ND or a HFD for 31 weeks prior to the time of sacrifice, from each group were dissected, weighed and snap frozen in liquid nitrogen. Homogenization was performed using a Potter-Elvehjem homogenizer (400 rpm, 10 strokes) in a sample solution containing 7 M urea, 2 M thiourea, 1% (w/v) dithiothreitol, 2% (w/v) CHAPS and 1.0% (v/v) IPG buffer 3-10. The samples were then sonicated for 2 × 4 s, shaken for 1 h at 30 °C and centrifuged (30 000 × g, 30 °C, 30 min). The clear infranatant was recovered and total protein was measured with 2-D Quant kit (Amersham biosciences). Samples were stored in aliquots at -80 °C until analysis. Samples were thawed for 30 min (30 °C, 1000 rpm) before isoelectric focusing (IEF). Immobiline DryStrips (11 and 17 cm, pH 3-10 NL, Bio-Rad) were used for IEF. Prior to IEF, each strip was rehydrated in 200 or 300 µL of rehydration solution containing 100 µg protein for analytical gels, 200-500 µg protein for preparative gels or 50 µg for Western blot analysis. The rehydration solution consisted of 8 M urea, 15 mM dithiothreitol, 0.5% (w/v) CHAPS and 1% (v/v) IPG buffer 3-10. Strips were allowed to rehydrate overnight under a layer of mineral oil at 20 °C and 50 V in a Protean IEF Cell (BioRad). Focusing was carried out at 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 1000-8000 V over 30 min, and then 8000 V for 35 kVh, or 25 kVh for 11 cm strips used in the western blot analysis, to reach steady state. Following IEF the strips were equilibrated for 15 min in a solution containing 6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS, 50 mM Tris-HCl pH 8.8 and 65 mM dithiothreitol. In a second step, the strips were equilibrated for an additional 15 min in the same solution except that dithiothreitol was replaced by 260 mM iodoacetamide. All second dimension runs were performed either in an Ettan Daltsix electrophoresis system (Amersham biosciences) or in an Ettan Dalttwelve electrophoresis system (Amersham bio1702
Journal of Proteome Research • Vol. 5, No. 7, 2006
Hansson et al.
sciences) according to the manufacturer’s recommendations (12.5%T, 0.8% C, continuous). All strips were sealed at the top of the second dimension gels with 0.5% agarose and run overnight until the tracking dye had reached the anodic end. Staining, Image Acquisition, and Statistics. Gels were stained with SYPRO Ruby protein gel stain (Bio-Rad) according to the manufacturer’s recommendations. Briefly, gels were fixed in 40% ethanol and 10% acetic acid for 3 h, stained overnight in SYPRO Ruby protein gel stain, washed in 10% methanol and 7% acetic acid for 30-60 min and scanned with a FLA 3000 gel scanner (Fuji film) at 50 µm resolution (532 nm, O580, F1000). The 2D-PAGE image computer analysis was carried out using the Phoretix 2D v2002.01 software package (Nonlinear dynamics). Spots were detected and matched automatically and then manually controlled. Background subtraction was performed using the method of average on boundary and relative volumes were calculated with normalization to total volume of spots present in the individual gel to correct for staining and loading differences. Only spots that were present in all gels were used for normalization. Duplicate samples were analyzed. If both samples produced quantifiable spots, then the mean value was used, and if only one of the two spots could be measured, then that observation was used in the calculations. If none was present, the data point was regarded as missing and set to zero. Statistical analyses were performed using the nonparametric Spearman’s Rho, McNemar’s, Kruskal-Wallis and MannWhitney U tests. Further details of the 2D-PAGE data analysis process are given in the Supporting Information. Identification and Characterization of Proteins in Spots. Preparative 2D-PAGE, spot excision, protein digestion and mass spectrometry were performed at the Swegene proteomics resource center in Lund (http://www.swegene.org/proteomics). Briefly, protein spots that appeared to be regulated based on the image analysis were excised either using an Ettan Spot handling workstation (Amersham Biosciences) or using pipet tips and transferred to original Eppendorf tubes. All following steps were handled using an Ettan Spot handling workstation (Amersham Biosciences). The gel pieces were destained with 50% acetonitrile, 25 mM NH4HCO3, and dried using a speed vac concentrator for 10-15 min. Digestion was performed overnight at 37 °C with 12.5 µg/mL trypsin (Promega) in 50 mM NH4HCO3. The trypsin solution was added in a volume sufficient to cover the gel piece (∼10 µL). The digestion was terminated and the peptides extracted by addition of 5% trifluoroacetic acid in 75% acetonitrile in an equal volume as the trypsin solution. Sample (0.5 µL) was added to the target plate (Waters) and allowed to dry, then equal volume (0.5 µL) of matrix solution (5 mg/mL R-cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile and 0.1% trifluoroacetic acid) and sample (0.5 µL) was eluted onto the plate and allowed to dry. Mass Spectrometry. Mass spectrometry was performed using a MALDI-TOF mass spectrometer (MALDI LR HT, Waters). Each spectrum represented up to 200 laser shots, depending on the signal-to-noise ratio. The resulting mass spectra were internally calibrated using the auto-digested tryptic mass values visible in all spectra. Calibrated spectra were processed and peaks extracted by the Piums software.20 Multiple searches were done using the automated Piums software.21 Database searching was also performed using the MASCOT 2.0.0 software. Since proteins were recovered from gels, carbamidomethylation was set as a fixed modification and methionine oxidation as a variable one. The peptide tolerance was set to 50-200 ppm in
Inflammatory Response in White Adipose Tissue
the Piums software and to 200 ppm in the MASCOT software. Only one trypsin missed cleavage was allowed. The protein identifications were considered to be confident when the expectancy score was below 0.001. Data from the identification process is presented in table S3 as supplement information. The average sequence coverage of the identified proteins was approximately 43%. The molecular mass and pI of the identified proteins were evaluated by analysis of the mobility of the corresponding protein spot in the 2D-PAGE images. Western Blot Analysis. Parametrial WAT from 13 to 15 months old female mice, fed either a ND or a HFD for 36-40 weeks prior to the time of sacrifice, were dissected and snap frozen in liquid nitrogen and then homogenized using a Potter-Elvehjem homogenizer (400 rpm, 10 strokes) in 0.25 M Sucrose, 1 mM EDTA, pH 7.0, 1 mM dithiothreitol, 20 µg/mL leupeptin, 10 µg/mL antipain and 1 µg/mL pepstatin A. The samples were centrifuged at 10 000 × g for 25 min at 4 °C. Aliquots of the infranatant and pellet fractions were collected and stored at -20 °C until analyzed further. Protein concentration was determined using BCA-assay (Pierce). Proteins were resolved by either 1D-PAGE or 2D-PAGE and electroblotted to nitrocellulose membranes. Detection of protein was accomplished using: a monoclonal rat anti human galectin-3 antibody (a kind gift from Dr. Hakon Leffler, Lund University, Sweden), a polyclonal goat anti mouse TNFR antibody (Santa Cruz Biotechnology), a polyclonal rabbit anti human phosphoIκB-R (ser32) antibody (Cell Signaling Technology), a monoclonal mouse anti rabbit glyceraldehyde-3-phosphate dehydrogenase antibody (Chemicon international), a polyclonal rabbit anti mouse mast cell protease 4 antibody (a kind gift from Dr. Lars Hellman, Uppsala University, Sweden) and a monoclonal mouse anti rat pyruvate carboxylase antibody (a kind gift from Dr. John Wallace, University of Adelaide, Australia). Monoclonal mouse anti chicken R-Tubulin antibody (Sigma) was used as loading control. Western blots were developed using a CCD camera (LAS 1000, Fuji Film). Real Time Quantitative PCR. Parametrial WAT from 13 to 15 months old female mice fed HFD for 36-40 weeks prior to the time of sacrifice was dissected and snap frozen in liquid nitrogen. For quantification of F4/80 mRNA 7 months old female mice fed ND or HFD for 12 weeks prior to the time of sacrifice were used. Total RNA was isolated and purified using RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer’s recommendations. RNA integrity was verified with agarose gel electrophoresis. Total RNA (1 µg) was treated with DNase I (DNase I amplification grade, Invitrogen) and then reversely transcribed using random hexamers (Amersham Biosciences) and SuperScriptTMII RNaseH reverse transcriptase (Invitrogen Life Technologies) according to the manufacturer’s recommendations. The mRNA levels of spot14 (Assays-ondemand, Mm00493680_s1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Assays-on-demand, Mm99999915_g1) and F4/80 (Assays-on-demand, Mm00802530_m1) were quantified using TaqMan real-time PCR with an ABI 7900 system (Applied Biosystems). As a control to normalize gene expression, ribosomal protein 29s was used in SYBR green chemistry (forward primer: 5′-GGA GTC ACC CAC GGA AGT-3′ and reverse primer: 5′-TCC ATT CAA GGT CGC TTA GTC-3′). Omitting reverse transcriptase in the reactions checked the absence of contamination by genomic DNA. Each sample was analyzed in duplicates. Immunohistochemistry. Parametrial WAT from 9 to 10 months old female mice, fed either a ND or a HFD for 24 weeks
research articles prior to the time of sacrifice, was fixed overnight in 4% buffered paraformaldehyde, pH 7.2, dehydrated in graded ethanols, and embedded in paraffin. Sections (6 µm) were mounted on slides and deparaffinized. The sections were incubated overnight at 4 °C with a monoclonal rat anti mouse F4/80 antibody (Serotec), diluted 1 to 200 in PBS (pH 7.2) containing 0.25% BSA and 0.25% Triton-X 100. After a rinsing step, sections were incubated with anti rat IgG coupled to Texas-Red (Jackson, West Grove, PA, US) for 1 h at room temperature. After a second rinsing step, sections were mounted in PBS/glycerol (1/1). Immunofluorescence was examined in an epifluorescence microscope (Olympus BX60) and images were captured with a digital camera (Olympus DP50). Cellular Fractionation. Periovarial WAT from 7 months old female mice, fed a ND or HFD for 12 weeks prior to the time of sacrifice, was excised, cut into small pieces and incubated in Krebs-Ringer solution (pH 7.4) supplemented with 3.5% BSA, 2 mM glucose, 200 nM adenosine and collagenase (1 mg/mL; Sigma) in a shaking incubator at 37 °C for ∼60 min, according to a modification22 of the Rodbell method.23 The digested tissue was filtered and the isolated cells were washed twice in Krebs-Ringer buffer (pH 7.4) with 1% BSA, 200 nM adenosine and 2 mM glucose by allowing the isolated adipocytes to float to the surface and then aspirate the underlying buffer. The aspirated buffer from the two washing steps was pooled and subjected to a brief centrifugation at 200 × g for 5 min, and the supernatant was removed, yielding a pellet containing the stromal-vascular fraction (SVF). Total RNA was isolated, purified, and analyzed as described above. Subcellular Fractionation. Parametrial WAT from 8 months old female mice fed HFD for 24 weeks prior to the time of sacrifice were dissected and homogenized using a PotterElvehjem homogenizer (400 rpm, 10 strokes) in 10 mM TrisHCl, pH 7.4, 2 mM EDTA, 250 mM Sucrose, 20 µg/mL leupeptin, 10 µg/mL antipain and 1 µg/mL pepstatin A. To remove cell debris and the fat cake, a brief centrifugation (5 min/80 g) was performed and the infranatant was removed using a syringe. The infranatant was then centrifuged at 720 g for 10 min and the resulting supernatant was discarded. The pellet was washed in a small volume of TES buffer and then centrifuged at 720 g for an additional 5 min. After removal of the supernatant the pellet, containing the nuclear fraction, was lysed in TES buffer supplemented with 5% SDS in a shaking incubator (Eppendorf) at 50 °C for 30 min and the protein concentration was determined using BCA-assay (Pierce). A 5-µg portion of protein from the nuclear fraction were separated using 1D-PAGE and the amount of GAPDH protein was measured using western blot analysis as described above from three individuals of each genotype. Retinyl Ester Hydrolase Activity. Parametrial WAT from 13 to 15 months old female mice fed ND or HFD for 36-40 weeks prior to the time of sacrifice were dissected and tissues were homogenized using a Potter-Elvehjem homogenizer (400 rpm, 10 strokes) in 0.25 M sucrose, 1 mM EDTA, pH 7.0, 1 mM dithiothreitol, 20 µg/mL leupeptin, 10 µg/mL antipain, and 1 µg/mL pepstatin A, followed by a centrifugation at 10 000 × g for 25 min at 4 °C. The fat cake was discarded and the infranatant was separated from the pellet. Retinyl ester hydrolase activity was measured using retinyl palmitate (RP) as substrate. The method is based on measurements of release of [14C] palmitate from retinyl-[14C]palmitate (American Radiolabeled Chemicals Inc.) and was adapted from previously described methods utilizing phospholipid-stabilized emulsions Journal of Proteome Research • Vol. 5, No. 7, 2006 1703
research articles
Figure 1. Protein level in WAT of mast cell protease 4 in 13-15 months old female mice, fed HFD for 36-40 weeks measured using 1D Western blot. Values given are mean fold change over control ( SEM, * indicates p < 0.05 analyzed with MannWhitney U test, n ) 4-5.
of triolein, diolein and cholesterol oleate.24 Briefly, [14C]RP (1 µCi) and unlabeled RP (together corresponding to 990 µg) was emulsified with 0.6 mg of phospholipids (phosphatidylcholine/ phosphatidylinositol, 3:1, w/w) in 20 mM KH2PO4 (pH 7.0), 1 mM EDTA, 1 mM dithioerythritol, 0.02% defatted BSA using sonication, to yield a final substrate concentration of 0.5 mM. This concentration has previously been shown to yield Vmax conditions,7 which was confirmed in our assay system. One unit of enzyme activity is equivalent to 1 µmol of fatty acids released/min at 37 °C. Specific activity is expressed as mU/mg of protein.
Results 1D-PAGE Protein Expression Profile of WAT from HSL-Null Mice Fed HFD. To compare the protein expression in WAT from HSL-null mice to that of wildtype littermates, proteins of crude homogenates were separated using 1D-PAGE. Following staining with coomassie brilliant blue, five differentially expressed proteins were identified using MALDI-TOF-MS. Two of these were found to have decreased expression levels, i.e., pyruvate carboxylase (PC) and polymerase I, and three increased expression levels, i.e., 94 kDa glucose-regulated protein (grp94), 78 kDa glucose-regulated protein and mast cell protease 4 (MCP-4), in HSL-null mice compared to wild-type littermates. The expression change for PC (0.15, p < 0.01) and MCP-4 (1.7, p < 0.05, Figure 1) was verified using 1D Western blot analysis. 2D-PAGE Protein Expression Profile of WAT from HSL-Null Mice Fed either ND or HFD. Having successfully identified five proteins with differential expression using 1D-PAGE separation, a more comprehensive protein expression profiling was performed using 2D-PAGE. In this analysis, duplicate gels from four individuals in each group were generated. A reference gel was created with 1026 matched spots. A representative gel together with enlargements of spots found to be differentially expressed is displayed in Figure 2. A series of Kruskal-Wallis tests were applied in order to identify the spots that had significant changes in their expression levels. In total, 297 spots were found to have a significantly changed expression level (p < 0.05). This is far more than the 50 spots, which are expected by chance alone on a 0.05-level. To be able to determine between which groups the differences were manifested, pairwise comparisons using Mann-Whitney U tests were also performed (p < 0.05). The number of differences found in the comparisons made was as follows: wild-type ND vs HSL-null ND (85 spots), wild-type HFD vs HSL-null HFD (140 spots), wild-type ND vs wild-type HFD (61 spots) and HSL-null ND vs HSL-null HFD (52 spots). The results indicate a larger influence 1704
Journal of Proteome Research • Vol. 5, No. 7, 2006
Hansson et al.
of the genotype than of the diet (85 and 140 vs 61 and 52 spots) on regulation of protein expression. Furthermore, they show that the expression differences between the two genotypes examined were more pronounced in the HFD groups than in the ND groups (140 spots vs 85 spots). Following identification of proteins with MALDI-TOF-MS, they were categorized according to their function (Table 1). In the category of metabolism two members of the acyl-CoA dehydrogenase protein family were found to be down-regulated in HSL-null mice compared with wild-type littermates in both diet groups studied, i.e., isovaleryl-CoA dehydrogenase (0.39 and 0.48, p < 0.05) and short chain acyl-CoA dehydrogenase (SCAD) (0.59 and 0.68, p < 0.05). SCAD was also found to be down-regulated in wild-type mice fed HFD in comparison with wild-type mice fed ND (0.84, p < 0.05). In connection to this finding, a decreased expression level of electron transferring flavoprotein alpha (ETFR) was observed in HSL-null mice compared with wild-type littermates in both diet groups studied (0.53 and 0.49, p < 0.05). Spot14, another protein involved in fatty acid metabolism, was detected in all groups examined with the exception of HSL-null mice fed HFD. The decrease in expression level of spot 14 in the HSL-null HFD group was verified using real time quantitative PCR (rtPCR) (0.31 HSL-null HFD vs wild-type HFD, p < 0.05). One exception to the observed down-regulation of proteins involved in fatty acid metabolism in HSL-null mice was found, i.e., ∆3,5,∆2,4-dienoyl-CoA isomerase (DI) (1.25 HSL-null ND vs wild-type ND, p < 0.05). DI is an auxiliary enzyme of unsaturated fatty acid β-oxidation. The largest difference found in the 2D-PAGE analysis was an upregulation of GAPDH. Two spots were identified as GAPDH, both being upregulated in HSL-null mice compared with wildtype littermates, regardless of diet (13.13, 6.92 and ON, ON, p < 0.05) (Table 1). Attempts were made to verify the change in expression with rtPCR and 1D Western blot analysis, but no significant increase in the expression of GAPDH was detected (data not shown), on the contrary a significant decrease was found at the mRNA level (0.55 HSL-null HFD vs wild-type HFD, p < 0.05). In a 2D Western blot analysis, a signal corresponding to the up-regulated spots identified as GAPDH in the 2D-PAGE protein expression profile were detected in samples corresponding to WAT from HSL-null mice from both diet groups investigated, but it was not detected in samples corresponding to WAT from wild-type mice (Figure 3). To investigate the localization of GAPDH a subcellular fractionation was performed of WAT from HSL-null mice and wild-type littermates fed HFD. Using 1D Western blot analysis, an approximately 10-fold increase in the amount of GAPDH was detected in the nuclear fraction of WAT from HSL-null mice compared to wildtype littermates (data not shown). Four proteins were categorized as being involved in inflammation, i.e., macrophagecapping protein g (capg), annexin A2, galectin-3 (gal-3), and ferritin heavy chain (ferritin H). Capg was found to have an increased expression level in WAT from HSL-null mice fed ND to the same level as wild-type mice fed HFD in comparison with wild-type mice fed ND (2.95 and 3.04 respectively, p < 0.05). Capg is an abundant protein in macrophages and is believed to be involved in actin function. Annexin A2 is expressed on the surface of macrophages and has been shown to be a binding site for plasminogen.25 It has also recently been reported that annexin A2 is a thiazolidinedione-responsive gene involved in glucose transporter 4 translocation in 3T3-L1 adipocytes.26 A reduction in the expression of annexin A2 was observed in HSL-null mice in both diet groups, compared with
Inflammatory Response in White Adipose Tissue
research articles
Figure 2. Representative SYPRO Ruby stained 2D-PAGE gel images of WAT. The total protein load was 100 µg. For separation in the first dimension a nonlinear pH 3-10 Immobiline DryStrip was used and for the second dimension a continuous 12.5% T, 0.8% C polyacrylamide gel was used. The gels display 1026 matched spots. Representative enlargements of spots found to be differentially expressed are presented.
wild-type littermates (0.31 and 0.35 respectively, p < 0.05). Gal-3 is an important mediator of inflammation and is expressed in activated macrophages. The spot identified as gal-3 was found in 2D-gels corresponding to WAT from HSL-null mice from both diet groups examined, but it was not detected in samples corresponding to WAT from wild-type mice. A tendency toward a higher expression level in HSL-null mice
fed HFD compared to HSL-null mice fed ND was detected, but this difference was not statistically significant. The increased expression level of gal-3 in HSL-null mice was confirmed (4.37, p < 0.001, HSL-null ND vs wild-type ND and 1.93, p < 0.05 HSL-null HFD vs wild-type HFD) using 1D Western blot analysis (Figure 4). An inflammatory response in wild-type mice fed HFD was also indicated by an increased protein level of Journal of Proteome Research • Vol. 5, No. 7, 2006 1705
research articles
Hansson et al.
Table 1. Differentially Expressed Proteins in WAT from HSL-Null Mice, Fed Either ND or HFD, Compared with Wild-Type Littermatesa ratio protein
Swiss Prot ac. no.
isovaleryl-CoA dehydrogenase SCAD ∆3,5,∆2,4-dienoyl-CoA isomerase spot14 aldehyde dehydrogenase ETF alpha GAPDH GAPDH carbonyl reductase 3 carbonic anhydrase III carbonic anhydrase III heme binding protein 1 ferritin light chain 1
Q9JHI5 Q07417 O35459 Q62264 P47738 Q99LC5 P16858 P16858 Q8K354 P16015 P16015 Q9R257 P29391
capg protein annexin A2 galectin-3 ferritin heavy chain
Q99LB4 P07356 P16110 P09528
chloride intracell. channel pr. 1 dual specificity pp 3 inorganic pyrophosphatase HSP60 actin, cytoplasmic 2 apolipoprotein A-I vimentin vimentin
Q9Z1Q5 Q9D7 × 3 Q9D819 P63038 P63260 Q00623 P20152 P20152
HSL-null ND vs wild-type ND
Metabolism 0.39 0.59 1.25 N. C. N. C. 0.53 ON 13.13 0.29 0.38 0.28 0.28 0.49 Inflammation 2.95 0.31 ON 8.44 Others 4.86 N. C. 0.36 0.53 3.78 2.66 10.78 0.36
HSL-null HFD vs wild-type HFD
wild-type HFD vs wild-type ND
HSL-null HFD vs HSL-null ND
N. C. 0.68 N. C. OFF 1.41 0.49 ON 6.92 N. C. N. C. N. C. N. C. 0.37
0.48 0.84 N. C. N. C. 0.63 N. C. s N. C. N. C. 0.57 0.32 N. C. N. C.
N. C. N. C. 0.52 OFF N. C. N. C. N. C. N. C. N. C. N. C. N. C. N. C. N. C.
N. C. 0.35 ON 2.74
3.04 N. C. s 3.17
N. C. N. C. N. C. N. C.
2.18 0.40 N. C. 0.52 3.33 N. C. OFF N. C.
2.76 N. C. 0.50 N. C. N. C. N. C. N. C. N. C.
N. C. N. C. N. C. N. C. N. C. N. C. OFF 4.18
a p < 0.05, Mann-Whitney U tests. No change (N. C.), not expressed, but expressed in the control group (OFF), expressed, but not in the control group (ON), not expressed in either group (-).
Figure 3. Representative 2D Western blots of glyceraldehyde3-phosphate dehydrogenase of WAT from wildtype mice fed ND (A) or HFD (B) and from HSL-null mice fed ND (C) or HFD (D). Arrows indicate the signals corresponding to the differentially expressed spots identified as GAPDH in the 2D-PAGE protein expression profile. n ) 2-3.
gal-3 in wild-type mice fed HFD compared with wild-type mice fed ND (2.69, p < 0.01). Another indication of an inflammatory response in WAT from HSL-null mice was a large increase in the expression level of ferritin H in WAT from HSL-null mice (8.44, p < 0.05, HSL-null ND vs wild-type ND and 2.74, p < 0.05 HSL-null HFD vs wild-type HFD). Ferritin is the major iron-binding protein in eukaryotic cells. It is composed of 24 subunits of ferritin H and ferritin light chain (ferritin L), assembled in various ratios in different tissues and disease states, including inflammation.27 Elevated serum level of ferritin is a characteristic of states of chronic inflammation and it has previously been shown that the expression of ferritin H can be 1706
Journal of Proteome Research • Vol. 5, No. 7, 2006
Figure 4. Protein level in WAT of galectin-3 (gal-3) in 13-15 months old female mice, fed either ND or HFD for 36-40 weeks measured using 1D Western blot. R-tubulin was used as an internal control, n ) 6-7. Values given are mean ( SEM, * indicates p < 0.05, ** indicates p < 0.01 and *** indicates p < 0.001 analyzed with Mann-Whitney U test.
induced by TNFR.28 Furthermore, an increase in the expression level of ferritin H was also detected when comparing WAT from wild-type mice fed HFD and wild-type mice fed ND (3.17, p < 0.05). A down-regulation of ferritin L was detected in WAT from HSL-null mice fed either ND or HFD compared with wild-type littermates (0.49, p < 0.05, HSL-null ND vs wild-type ND and 0.37, p < 0.05 HSL-null HFD vs wild-type HFD). No significant difference in the expression of ferritin L was detected in WAT from wild-type mice fed HFD compared with wild-type mice fed ND. Aldehyde dehydrogenase is an enzyme involved in the conversion of retinol to retinoic acid. The expression level of the enzyme was found to be increased in HSL-null mice fed HFD in comparison with wild-type mice fed HFD (1.41, p < 0.05), whereas a decreased level was observed when comparing
Inflammatory Response in White Adipose Tissue
research articles
Figure 5. Protein level in WAT of TNFR in 13-15 months old female mice, fed HFD for 36-40 weeks measured using 1D Western blot. TNFR of 17 kDa corresponds to the released soluble form and TNFR of 26 kDa corresponds to the membrane-bound form. n ) 4-5, values given are mean fold change over control ( SEM, * indicates p < 0.05 analyzed with Mann-Whitney U test.
Figure 7. Immunohistochemistry images of epididymal WAT from 9 to 10 months old wild-type mice fed ND (A) or HFD (B) and from HSL-null mice fed ND (C) or HFD (D) for 24 weeks. Increased staining for the specific macrophage marker F4/80 (red) indicates a larger infiltration of this cell type in WAT from HSLnull mice in both diet groups investigated compared with wildtype littermates. Figure 6. Phosphorylation level of IκB in WAT from 13 to 15 months old female mice, fed HFD for 36-40 weeks, analyzed using 1D Western blot and phosphospecific antibodies (Ser 32). R-tubulin was used as an internal control, n ) 4-5. Values given are mean fold change over control ( SEM, * indicates p < 0.05 analyzed with Mann-Whitney U test.
wild-type mice fed HFD with wild-type mice fed ND (0.63, p < 0.05). Increased Level of TNFR in WAT from HSL-Null Mice Fed HFD. As the protein expression profile indicated an increased expression of inflammation-related proteins, i.e., ferritin H and gal-3 among others, the level of TNFR was investigated in WAT from HSL-null mice and wild-type littermates fed HFD using 1D Western blot analysis. TNFR is an important cytokine and mediator of inflammation and the protein level gives an indication of the inflammatory status of the animals. TNFR consists of a membrane bound 26 kDa protein, which can be cleaved to a soluble and secreted 17 kDa form.29 In WAT from HSL-null mice fed HFD a severalfold increase in the levels of both isoforms was observed, 4.03 p < 0.01 and 3.51 p < 0.01, respectively (Figure 5). This increase of TNFR expression is in agreement with previous findings where an increase of TNFR mRNA level has been reported in HSL-null mice fed either a ND or a HFD.12 Increased Activation of NFκB in WAT from HSL-Null Mice Fed HFD. Nuclear factor kappa B (NFκB) is a nuclear transcription factor regulating the expression of many proinflammatory genes, including TNFR. Activation of NFκB was measured indirectly by measuring the phosphorylation level of IκB-R, an inhibitor of NFκB that is inactivated upon phosphorylation of Ser-32. Using 1D Western blot analysis and a phosphospecific antibody, a severalfold increase in phosphorylation at Ser-32 of IκB-R was observed (3.47, p < 0.01) in WAT from HSL-null mice fed HFD compared with wild-type littermates (Figure 6). Macrophage Infiltration of WAT from HSL-Null Mice Fed either ND or HFD. To assess the degree of macrophage
Figure 8. mRNA level of the macrophage marker F4/80 in the stromal-vascular fraction of WAT from 7 months old female mice, fed ND or HFD for 12 weeks, analyzed with rtPCR. n ) 4-5. Values given are mean ( SEM, * indicates p < 0.05 analyzed with Mann-Whitney U test.
infiltration in WAT, immunohistochemistry was performed using antibodies against F4/80, a commonly used and specific macrophage marker. An increased F4/80 signal in sections of WAT from HSL-null mice was detected compared to wild-type littermates (Figure 7). This sign of increased infiltration of macrophages was found in both the ND and the HFD group, indicating increased inflammation in WAT of HSL-null mice regardless of diet. The mRNA level of F4/80 was also investigated using rtPCR. To investigate the location of the F4/80 positive cells, WAT was fractionated in an adipocyte fraction and a SVF prior to the rtPCR analysis. The SVF contained a larger amount of F4/80 mRNA then the adipocyte fraction, irrespective of either genotype or diet (data not shown). A significant increase in SVF of F4/80 mRNA was observed when comparing wild-type mice fed HFD with wild-type mice fed ND (2.10, p < 0.05) (Figure 8). Furthermore, the SVF from HSLnull mice contained larger amounts of F4/80 mRNA compared with the corresponding fraction from wild-type littermates in both the ND (4.29, p < 0.05) and the HFD (4.70, p < 0.05) group. Journal of Proteome Research • Vol. 5, No. 7, 2006 1707
research articles
Figure 9. Total retinyl ester hydrolase activity in homogenates of WAT from 13 to 15 months old female mice, fed either ND or HFD for 36-40 weeks. n ) 6-7. Values given are mean ( SEM, * indicates p < 0.05 and ** indicates p < 0.01 analyzed with Mann-Whitney U test.
This was also observed in the adipocyte fraction (data not shown). Decreased Retinyl Ester Hydrolase Activity in WAT from HSL-Null Mice Fed either ND or HFD. To test the hypothesis that the inflammatory response in WAT of HSL-null mice is due to a reduced ability to generate retinoic acid, which has antiinflammatory properties,30 from retinyl ester stores the retinyl ester hydrolase activity was investigated. A significant decrease in total retinyl ester hydrolase activity was found in homogenates of WAT from HSL null mice fed either ND (0.23, p < 0.01) or HFD (0.23, p < 0.01) compared to wild-type littermates. A significant decrease was also observed when comparing wild-type mice fed HFD with wild-type mice fed ND (0.64, p < 0.05) (Figure 9).
Discussion The HSL-null mouse model investigated here exhibits insulin resistance at several sites, including adipose tissue, liver, and skeletal muscle11 and is protected against diet-induced obesity (manuscript in preparation). It has previously been reported that the mRNA level of TNFR is increased in WAT from HSLnull mice,12 suggesting an inflammatory response in the tissue. To further characterize the inflammatory state of this mouse model, we chose to investigate the protein expression profile of WAT using 1D- and 2D-PAGE. In the protein expression profile, several proteins involved in fatty acid metabolism, in particular β-oxidation, were observed to have decreased expression in the HSL-null mice. Among the proteins found to have reduced expression were SCAD, ETFR, and Spot14. SCAD is involved in β-oxidation of fatty acids and ETFR is a protein that accepts electrons from the β-oxidation pathway and delivers them to the mitochondrial electron transport chain. Spot14 is a protein highly expressed in triglyceride synthesizing tissues like liver, WAT, and BAT and is believed to play a role in lipogenesis. On one hand, this finding indicates a decreased capacity of HSL-null mice to perform β-oxidation in WAT. On the other hand, out of several acyl-CoA dehydrogenases involved in β-oxidation SCAD was the only one found in the protein expression profile and SCAD is only responsible for degradation of short chain fatty acids (C4 and C6). Further experiments are needed in order to clarify this aspect of the phenotype. The largest increase in expression found in the 2D-PAGE analysis was an up-regulation of GAPDH. The molecular weight of the increased protein was, however, approximately 3-5 kDa lower than the expected size of 36 kDa. When attempting to 1708
Journal of Proteome Research • Vol. 5, No. 7, 2006
Hansson et al.
verify this change in expression with 2D Western blot analysis both the full-length GAPDH and a smaller isoform were detected in WAT from HSL-null mice, but only the full-length isoform was detected in WAT from wild-type mice. The nature of the smaller GAPDH variant is not known but there are several possibilities. It could represent a splice variant, a post-translationally modified variant or a degradation product of the fulllength protein. Although protein degradation may be the most obvious explanation, the fact that peptides from both the C-terminal and N-terminal end of the protein were represented in the MALDI-TOF-MS identification of the protein argues against this. GAPDH is known as a glycolytic enzyme, but in recent years, it has been demonstrated that GAPDH is involved in a number of diverse functions including apoptosis.31,32 Many of these new functions include a translocation of the protein to the nuclear compartment. The data presented here indicates that a larger amount of GAPDH is localized in the nucleus in HSL null mice compared with wild-type littermates. However, at this point we do not know if this corresponds to the smaller isoform or not. It has previously been shown that a truncation or mutation of a highly charged 13 amino acid nuclear export signal (NES) peptide in GAPDH results in a nuclear localization of the protein.33 A perturbation of this NES could be an explanation for the data presented concerning GAPDH. The observations made concerning GAPDH in the 2D-PAGE analysis highlights an important issue when interpreting data obtained with this technique. The observed expression changes may be a result of posttranslational modifications of the protein that change its properties without affecting the total expression level. It is possible that other isoforms of the same protein display changed expression without being identified in the analysis. Because of this problem care should be taken before making any assumptions regarding the biological importance of such changes before appropriate verification experiments have been made. The present study demonstrates the presence of an inflammatory response in WAT from the nonobese HSL-null mouse model in both diet groups investigated. There are several manifestations of this inflammatory response, including increased protein levels of several well-known markers of inflammation, like TNFR and ferritin H, activation of NFκB, increased expression of several macrophage and mast cell proteins, including gal-3, capg, and MCP-4, and macrophage infiltration in WAT. To determine at what age the inflammatory response first is manifested more experiments are clearly needed. However, as the animals used in the fractionation experiment were 7 months old the inflammatory response is present at least from this age. Many factors have been implicated to provide the link between obesity and inflammation in WAT. Insulin is known to have antiinflammatory effects and in insulin-resistant states these effects are perturbed. As the HSL-null mouse model investigated here displays signs of insulin resistance at the level of WAT and elevated plasma insulin levels,11 it is possible that this factor contributes to the described inflammatory response. Other factors to take into consideration are adipokines, such as adiponectin (Acrp30). Acrp30 is a 30-kDa protein secreted from adipose tissue and decreased plasma level of this adipokine is normally observed in states of obesity. In mouse models, where the level of Acrp30 is manipulated, a lowering of the expression level of Acrp30 is usually accompanied by an increased level of TNFR. One example is the Acrp30-null mouse.34 Decreased WAT mRNA level of Acrp30 has previously
research articles
Inflammatory Response in White Adipose Tissue
been reported in HSL-null mice fed either ND or HFD12 and in the HSL-null mouse strain investigated here, decreased plasma levels of Acrp30 protein have been observed in both diet groups investigated (manuscript in preparation). One possible explanation for the observed increase of TNFR in WAT of HSL-null mice could be the decreased level of Acrp30. The inflammatory response in WAT is presumably the cumulative result of many different events, rather than induced by a single factor. A key question with regard to our model is how the absence of the metabolic enzyme HSL triggers WAT inflammation. At this point, we have no clear answer to this question. However, one could speculate that a connection between HSL expression and the regulation of target gene expression could be that HSL provides ligands for transcription factors such as PPARγ or any of the retinoid nuclear receptors. Keeping the broad substrate specificity of HSL in mind this ligand could be any of a number of candidates. An appealing candidate is some derivate of vitamin A, i.e., an isomer of retinoic acid (RA). Isomers of RA have previously been shown to exert anti-inflammatory and immunomodulatory effects.30 Furthermore, a reduced or absent vitamin A signal leads to activation of NFκB, thereby possibly providing a link between vitamin A and an inflammatory response in WAT.35 The antiinflammatory role of RA is further supported by findings of its inhibitory effect on inflammatory cytokine production in an activated macrophage cell line.36 Among the cytokines reported to be inhibited by RA are nitric oxide, interleukin-1 beta and TNF-R.36 Gal-3 expression level has been shown to be suppressed by RA in a F9 cell line,37 thereby providing further support for an anti-inflammatory role of RA. It has previously been shown that adipocytes contain a significant amount of the total body store of retinyl esters. In fact, in rats, adipose tissue is the second largest depot after the liver and contains approximately 15% of total retinyl esters present in the body.38 However, the mechanism behind the mobilization of these stores has not yet been fully elucidated. It has been suggested that HSL may play a role in this mobilization of retinyl ester stores in WAT.7 The reduction of retinyl ester hydrolase activity in WAT from HSL-null mice (Figure 9), together with increased retinyl ester stores (manuscript in preparation), observed in our HSL-null mice clearly indicates that HSL could have this function in WAT. In 2002, Balmer and Blomhoff published a summary of 532 genes known to be regulated by RA.39 Among these are several of the proteins found to have a differential expression in the protein expression profile presented here, including spot14, apolipoprotein A1, gal-3, aldehyde dehydrogenase, grp94, and vimentin, providing further support for the hypothesis that HSL plays a role in RA-mediated gene regulation. Our working hypothesis is that in the absence of HSL in adipocytes, and possibly other cell types present in WAT, such as preadipocytes, epithelial cells, or macrophages, a RA ligand is not generated to suppress NFκB activity. The increased activity of NFκB then leads to an increased level of TNFR in WAT. The possible decrease of RA in macrophages could also give rise to increased levels of other pro-inflammatory molecules like nitric oxide and interleukin-1 beta. Furthermore, gal-3 is known to be involved in chemotaxis, i.e., attracting inflammatory cells to sites of inflammation and thereby contributing to the recruitment of macrophages and other inflammatory cells to WAT. Many important questions still remain unanswered. One such is if RA has a role in the inflammatory
response in WAT in a normal setting, keeping in mind the extreme genotype that a null mouse model represents. In conclusion, to analyze the data generated by 2D-PAGE we have used measurements of the correlation between means and standard deviation to evaluate different parts of the data analysis process and also presented a way to deal with the problem of missing data by setting up a few simplistic rules. The present study has also demonstrated the presence of an inflammatory response in WAT from the nonobese HSL-null mouse model, fed either a ND or a HFD. During the preparation of this manuscript, Cinti and coworkers have demonstrated a 15-fold increase in necrotic-like adipocyte death in an independently generated HSL-null mouse strain fed normal chow diet. In agreement with the data presented here, they also show increased macrophage infiltration and increased expression of gal-3 and TNF-R.40
Acknowledgment. We thank Ann-Helen Thore´n for animal breeding and genotyping, Dr. Hakon Leffler, Dr. Lars Hellman, Dr. John Wallace and Dr. Patrik Brundin for their kind gifts of antibodies. Financial support was provided by the Swedish Research Council (project no. 11284 to C.H. and project no. 4499 to F.S.), Cell Factory for Functional Genomics, a program funded by the Swedish Foundation for Strategic Research, Center of Excellence Grant from the Juvenile Diabetes Foundation, USA, and Knut and Alice Wallenberg Foundation, Sweden, the Swedish Diabetes Association and the following foundations: Novo Nordisk, Denmark, A. Påhlsson, Salubrin/ Druvan and Torsten and Ragnar So¨derberg. Supporting Information Available: Evaluation of the data analysis process in the 2D-PAGE experiment and two supporting information tables (Tables S1 and S2). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Holm, C.; Belfrage, P.Fredrikson, G. Immunological evidence for the presence of hormone-sensitive lipase in rat tissues other than adipose tissue. Biochem. Biophys. Res. Commun. 1987, 148, 99105. (2) Holm, C.; Fredrikson, G.; Cannon, B.Belfrage, P. Hormonesensitive lipase in brown adipose tissue: identification and effect of cold exposure. Biosci. Rep. 1987, 7, 897-904. (3) Mulder, H.; Holst, L. S.; Svensson, H.; Degerman, E.; Sundler, F.; Ahren, B.; Rorsman, P.Holm, C. Hormone-sensitive lipase, the rate-limiting enzyme in triglyceride hydrolysis, is expressed and active in beta-cells. Diabetes 1999, 48, 228-232. (4) Holst, L. S.; Langin, D.; Mulder, H.; Laurell, H.; Grober, J.; Bergh, A.; Mohrenweiser, H. W.; Edgren, G.Holm, C. Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase. Genomics 1996, 35, 441-447. (5) Fredrikson, G.; Stralfors, P.; Nilsson, N. O.Belfrage, P. Hormonesensitive lipase of rat adipose tissue. Purification and some properties. J. Biol. Chem. 1981, 256, 6311-6320. (6) Lee, F. T.; Adams, J. B.; Garton, A. J.Yeaman, S. J. Hormonesensitive lipase is involved in the hydrolysis of lipoidal derivatives of estrogens and other steroid hormones. Biochim. Biophys. Acta 1988, 963, 258-264. (7) Wei, S.; Lai, K.; Patel, S.; Piantedosi, R.; Shen, H.; Colantuoni, V.; Kraemer, F. B.Blaner, W. S. Retinyl ester hydrolysis and retinol efflux from BFC-1beta adipocytes. J. Biol. Chem. 1997, 272, 14159-14165. (8) Osuga, J.; Ishibashi, S.; Oka, T.; Yagyu, H.; Tozawa, R.; Fujimoto, A.; Shionoiri, F.; Yahagi, N.; Kraemer, F. B.; Tsutsumi, O.Yamada, N. Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 787-792. (9) 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. J. Biol. Chem. 2002, 277, 4806-4815.
Journal of Proteome Research • Vol. 5, No. 7, 2006 1709
research articles (10) Wang, S. P.; Laurin, N.; Himms-Hagen, J.; Rudnicki, M. A.; Levy, E.; Robert, M. F.; Pan, L.; Oligny, L.Mitchell, G. A. The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice. Obes Res. 2001, 9, 119-128. (11) Mulder, H.; Sorhede-Winzell, M.; Contreras, J. A.; Fex, M.; Strom, K.; Ploug, T.; Galbo, H.; Arner, P.; Lundberg, C.; Sundler, F.; Ahren, B.Holm, C. Hormone-sensitive lipase null mice exhibit signs of impaired insulin sensitivity whereas insulin secretion is intact. J. Biol. Chem. 2003, 278, 36380-36388. (12) Harada, K.; Shen, W. J.; Patel, S.; Natu, V.; Wang, J.; Osuga, J.; Ishibashi, S.Kraemer, F. B. Resistance to high-fat diet-induced obesity and altered expression of adipose-specific genes in HSLdeficient mice. Am. J. Physiol. Endocrinol. Metab. 2003, 285, E1182-1195. (13) Park, S. Y.; Kim, H. J.; Wang, S.; Higashimori, T.; Dong, J.; Kim, Y. J.; Cline, G.; Li, H.; Prentki, M.; Shulman, G. I.; Mitchell, G. A.Kim, J. K. Hormone-Sensitive Lipase Knockout Mice Have Increased Hepatic Insulin Sensitivity and Are Protected from Short-Term Diet-Induced Insulin Resistance in Skeletal Muscle and Heart. Am. J. Physiol. Endocrinol. Metab. 2005. (14) Dandona, P.; Aljada, A.Bandyopadhyay, A. Inflammation: the link between insulin resistance, obesity and diabetes. Trends Immunol. 2004, 25, 4-7. (15) Fried, S. K.; Bunkin, D. A.Greenberg, A. S. Omental and subcutaneous adipose tissues of obese subjects release interleukin-6: depot difference and regulation by glucocorticoid. J. Clin. Endocrinol. Metab. 1998, 83, 847-850. (16) Samad, F.; Yamamoto, K.; Pandey, M.Loskutoff, D. J. Elevated expression of transforming growth factor-beta in adipose tissue from obese mice. Mol. Med. 1997, 3, 37-48. (17) Hotamisligil, G. S.; Shargill, N. S.Spiegelman, B. M. Adipose expression of tumor necrosis factor-alpha: direct role in obesitylinked insulin resistance. Science. 1993, 259, 87-91. (18) Weisberg, S. P.; McCann, D.; Desai, M.; Rosenbaum, M.; Leibel, R. L.Ferrante, A. W., Jr. Obesity is associated with macrophage accumulation in adipose tissue. J. Clin. Invest. 2003, 112, 17961808. (19) Neuhoff, V.; Arold, N.; Taube, D.; Ehrhardt, W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis. 1988, 9, 255-262. (20) Samuelsson, J.; Dalevi, D.; Levander, F.; Rognvaldsson, T. Modular, scriptable and automated analysis tools for high-throughput peptide mass fingerprinting. Bioinformatics 2004, 20, 3628-3635. (21) Levander, F.; Rognvaldsson, T.; Samuelsson, J.; James, P. Automated methods for improved protein identification by peptide mass fingerprinting. Proteomics 2004, 4, 2594-2601. (22) Honnor, R. C.; Dhillon, G. S.; Londos, C. cAMP-dependent protein kinase and lipolysis in rat adipocytes. I. Cell preparation, manipulation, and predictability in behavior. J. Biol. Chem. 1985, 260, 15122-15129. (23) Rodbell, M. Metabolism of Isolated Fat Cells. I. Effects of Hormones on Glucose Metabolism and Lipolysis. J. Biol. Chem. 1964, 239, 375-80. (24) Osterlund, T.; Danielsson, B.; Degerman, E.; Contreras, J. A.; Edgren, G.; Davis, R. C.; Schotz, M. C.; Holm, C. Domain-structure analysis of recombinant rat hormone-sensitive lipase. Biochem. J. 1996, 319 (Pt 2), 411-420. (25) Falcone, D. J.; Borth, W.; Khan, K. M.; Hajjar, K. A. Plasminogenmediated matrix invasion and degradation by macrophages is
1710
Journal of Proteome Research • Vol. 5, No. 7, 2006
Hansson et al.
(26)
(27) (28)
(29)
(30) (31) (32) (33)
(34)
(35)
(36) (37)
(38)
(39) (40)
dependent on surface expression of annexin II. Blood 2001, 97, 777-784. Huang, J.; Hsia, S. H.; Imamura, T.; Usui, I.; Olefsky, J. M. Annexin II is a thiazolidinedione-responsive gene involved in insulininduced glucose transporter isoform 4 translocation in 3T3-L1 adipocytes. Endocrinology 2004, 145, 1579-1586. Harrison, P. M.; Arosio, P. The ferritins: molecular properties, iron storage function and cellular regulation. Biochim. Biophys. Acta 1996, 1275, 161-203. Torti, S. V.; Kwak, E. L.; Miller, S. C.; Miller, L. L.; Ringold, G. M.; Myambo, K. B.; Young, A. P.; Torti, F. M. The molecular cloning and characterization of murine ferritin heavy chain, a tumor necrosis factor-inducible gene. J. Biol. Chem. 1988, 263, 1263812644. Xu, H.; Uysal, K. T.; Becherer, J. D.; Arner, P.; Hotamisligil, G. S. Altered tumor necrosis factor-alpha (TNF-alpha) processing in adipocytes and increased expression of transmembrane TNFalpha in obesity. Diabetes 2002, 51, 1876-1883. Stephensen, C. B. Vitamin A, infection, and immune function. Annu. Rev. Nutr. 2001, 21, 167-192. Berry, M. D.; Boulton, A. A. Glyceraldehyde-3-phosphate dehydrogenase and apoptosis. J. Neurosci. Res. 2000, 60, 150-154. Sirover, M. A. New nuclear functions of the glycolytic protein, glyceraldehyde-3-phosphate dehydrogenase, in mammalian cells. J. Cell. Biochem. 2005, 95, 45-52. Brown, V. M.; Krynetski, E. Y.; Krynetskaia, N. F.; Grieger, D.; Mukatira, S. T.; Murti, K. G.; Slaughter, C. A.; Park, H. W.; Evans, W. E. A novel CRM1-mediated nuclear export signal governs nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase following genotoxic stress. J. Biol. Chem. 2004, 279, 59845992. Maeda, N.; Shimomura, I.; Kishida, K.; Nishizawa, H.; Matsuda, M.; Nagaretani, H.; Furuyama, N.; Kondo, H.; Takahashi, M.; Arita, Y.; Komuro, R.; Ouchi, N.; Kihara, S.; Tochino, Y.; Okutomi, K.; Horie, M.; Takeda, S.; Aoyama, T.; Funahashi, T.; Matsuzawa, Y. Diet-induced insulin resistance in mice lacking adiponectin/ ACRP30. Nat Med. 2002, 8, 731-737. Carlsen, H.; Alexander, G.; Austenaa, L. M.; Ebihara, K.; Blomhoff, R. Molecular imaging of the transcription factor NF-kappaB, a primary regulator of stress response. Mutat. Res. 2004, 551, 199211. Mathew, J. S.Sharma, R. P. Effect of all-trans-retinoic acid on cytokine production in a murine macrophage cell line. Int. J. Immunopharmacol. 2000, 22, 693-706. Lu, Y.; Amos, B.; Cruise, E.; Lotan, D.Lotan, R. A parallel association between differentiation and induction of galectin-1, and inhibition of galectin-3 by retinoic acid in mouse embryonal carcinoma F9 cells. Biol. Chem. 1998, 379, 1323-1331. Tsutsumi, C.; Okuno, M.; Tannous, L.; Piantedosi, R.; Allan, M.; Goodman, D. S.; Blaner, W. S. Retinoids and retinoid-binding protein expression in rat adipocytes. J. Biol. Chem. 1992, 267, 1805-1810. Balmer, J. E.Blomhoff, R. Gene expression regulation by retinoic acid. J. Lipid Res. 2002, 43, 1773-1808. Cinti, S.; Mitchell, G.; Barbatelli, G.; Murano, I.; Ceresi, E.; Faloia, E.; Wang, S.; Greenberg, A. S.; Obin, M. S. Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans. J. Lipid Res. 2005.
PR060101H
