Iron Inhibits the Secretion of Apolipoprotein E in Cultured Human Adipocytes

Abstract

Nonalcoholic steatohepatitis (NASH) is characterized by adipose tissue dysfunction with insulin resistance and the dysregulation of adipokines.1Hebbard L. et al.Nat Rev Gastroenterol Hepatol. 2011; 8: 35-44Crossref PubMed Scopus (348) Google Scholar Recent data indicate repartitioning of iron from the liver to adipocytes in obesity and a role for iron in the development of adipose tissue dysfunction.2Orr J.S. et al.Diabetes. 2014; 63: 421-432Crossref PubMed Scopus (108) Google Scholar, 3Simcox J.A. et al.Cell Metab. 2013; : 329-341Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar However, the molecular mechanisms have not been established. To test the hypothesis that iron modulates adipokine release, we performed a quantitative proteomics analysis of the human Simpson-Golabi-Behmel Syndrome (SGBS) adipocyte secretome after 48 hours of treatment with ferric ammonium citrate (FAC). We used stable isotope-labeled amino acids in cell culture (SILAC) to characterize changes in the adipocyte secretome in response to iron. This technique has enabled direct comparison of quantities of individual proteins in the adipocyte secretome in response to iron using a proteomics approach as a tool for the identification of novel treatment targets in NASH. Detailed methodology is described in Supplementary Methods. We first showed that 100 μmol/L FAC causes significant adipocyte iron loading without compromising cell viability. We found that compared with vehicle, both 100 μmol/L and 500 μmol/L FAC caused significant increases in cellular iron concentration (P = .007 and P = .006, respectively) (Supplementary Figure 1A). There was no effect of iron loading on cellular viability (MTS) assay, total messenger RNA (mRNA), total whole-cell lysate protein, or total secretome protein (Supplementary Figure 1B–E). Given these findings, we selected 100 μmol/L FAC as the concentration to compare with vehicle in the secretome SILAC proteomic analysis. A total of 338 proteins were quantified in the adipocyte secretome by SILAC proteomics. These are represented by the volcano plot in Supplementary Figure 2 and the proteomics data have been deposited into the ProteomeXchange Consortium via the Proteomics Identifications (PRIDE) partner repository (www.proteomexchange.org) with the data set identifier PXD006341. Iron treatment led to significant differential secretion of 60 of these proteins (>2-fold change; P < .05). We then manually reviewed UniProt database descriptions of these 60 proteins.4The UniProt ConsortiumNucleic Acids Res. 2017; : D158-D169Crossref PubMed Scopus (3135) Google Scholar This generated a list of 20 proteins of interest (highlighted in bold in Supplementary Table 1). These proteins of interest and their synonyms then were entered into a PubMed title/abstract search in association with NASH and its synonyms. This identified 3 proteins as candidate intermediates for iron-induced adipose tissue dysfunction in NASH. These proteins were adiponectin, annexin A1, and apolipoprotein E (ApoE). Our SILAC analysis showed that iron treatment resulted in an 81% reduction in annexin A1 secretome signal intensity (P = .001). This may be important because annexinA1 knockout (KO) mice show greater degrees of hepatic lobular inflammation and fibrosis than controls when fed a methionine-choline–deficient diet.5Locatelli I. et al.Hepatology. 2014; 60: 531-544Crossref PubMed Scopus (75) Google Scholar Adipocyte iron also previously has been shown to transcriptionally down-regulate serum adiponectin in mouse-derived adipocytes, 3T3-L1 cells.6Gabrielsen J.S. et al.J Clin Invest. 2012; 122: 3529-3540Crossref PubMed Scopus (214) Google Scholar Our findings now support this in a human adipocyte cell line with a 55% reduction in adiponectin signal intensity in iron-treated SGBS cells (P = .005). We next focused on the iron regulation of ApoE secretion. ApoE appears to protect against steatohepatitis in mice. In an ApoE KO model, unlike wild-type controls, ApoE KO mice fed 7 weeks of a Western diet developed impaired glucose tolerance, steatohepatitis, and hepatic fibrosis.7Schierwagen R. et al.Sci Rep. 2015; 5: 12931Crossref PubMed Scopus (100) Google Scholar ApoE is a component of lipoproteins, and promotes very low density lipoprotein–induced adipogenesis.8Pendse A.A. et al.J Lipid Res. 2009; 50: S178-S182Crossref PubMed Scopus (116) Google Scholar ApoE knockout mice also readily develop atherosclerosis on an atherogenic diet.8Pendse A.A. et al.J Lipid Res. 2009; 50: S178-S182Crossref PubMed Scopus (116) Google Scholar Iron reduced secreted ApoE by 58% (P = .001) and 76% (P = .007), as measured by SILAC and Western blot, respectively. Conversely, iron treatment increased intracellular ApoE levels by more than 11-fold (P = .0005), without causing a significant change in mRNA levels (Figure 1). It therefore appears that iron inhibits the secretion of ApoE from adipocytes, causing ApoE to become sequestered intracellularly. Similar effects on ApoE secretion have been shown with iron treatment in primary cultured astrocytes and cortical neurons.9Xu H. et al.J Alzheimers Dis. 2016; 31: 471-487Crossref Scopus (29) Google Scholar Taken together with our data, it seems possible that iron may have similar effects on a range of cell types and represents a clear target for further investigation. Treatment with iron in our study showed an up-regulation of anti-oxidant responses (heme-oxygenase-1 and glutathione peroxidase-1 mRNA), indicating the presence of oxidative stress. Interleukin 6 mRNA, however, was not increased with iron treatment, and there was no difference among multiple markers of endoplasmic reticulum stress (Supplementary Figure 3A–I). We considered whether iron may have a generalized effect on pathways of protein secretion, used by a variety of proteins. We evaluated the role of iron in the secretion of proteins by the classic and exosomal pathways using the UniProt and EVpedia databases, respectively.4The UniProt ConsortiumNucleic Acids Res. 2017; : D158-D169Crossref PubMed Scopus (3135) Google Scholar, 10Kim D.K. et al.Bioinformatics. 2015; 31: 933-939Crossref PubMed Scopus (258) Google Scholar We found enrichment of signal peptide-containing (P = .02), but not exosome-secreted, proteins (P = .51) among the iron-dysregulated proteins, suggesting that iron may have a specific effect on proteins secreted via the classic pathway (Supplementary Figure 3J–M). This research has characterized the effect of iron on the adipocyte secretome. These data provide a platform for multiple avenues for future research. In addition, we have been able to show that increased iron results in sequestration of ApoE within adipocytes, which may be of key importance in the regulation of insulin resistance and liver injury in NASH. Identifying the molecular mechanisms of iron-induced inhibition of ApoE secretion from adipocytes, particularly relating to the role of oxidative stress, may show novel therapeutic strategies for improving adipocyte function in NASH. ProteomeXchange Consortium via the Proteomics Identifications (PRIDE) partner repository: http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=pxd006341 data set identifier PXD006341. SGBS pre-adipocytes were a gift from Martin Wabitsch (University of Ulm, Ulm, Germany).1Wabitsch M. et al.Int J Obes Relat Metab Disord. 2001; 25: 8-15Crossref PubMed Scopus (416) Google Scholar, 2Fischer-Posovszky P. et al.Obes Facts. 2008; : 184-189Crossref PubMed Scopus (190) Google Scholar SGBS cells were passaged, proliferated, and differentiated at less than 50 generations in 12-well plates and 100-mm dishes as previously described.3Luo X. et al.Diabetes. 2012; 61: 124-136Crossref PubMed Scopus (53) Google Scholar Cells were treated with 90 μg/mL heparin and 1 ng/mL fibroblast growth factor-1 (both Sigma-Aldrich, St. Louis, MO) throughout proliferation and differentiation. After 14 days of differentiation, cells were incubated with 0, 25, 100, or 500 μmol/L FAC (Sigma-Aldrich) for 24 hours. Media then was replaced with the same for a further 24 hours until the end of the experiment. RNA was extracted from treated SGBS adipocytes using a PureLink RNA mini kit (Invitrogen, Carlsbad, CA). Complementary DNA was synthesized from 1 μg RNA using a Sensifast complementary DNA synthesis kit (Bioline, London, UK) after treatment with DNase 1 (Invitrogen). Samples underwent thermal cycling using a ViiA7 real-time polymerase chain reaction machine (Invitrogen) with a Sensifast SYBR Lo-ROX Kit (Bioline). The following protocol was used: 2 minutes at 95°C, then 40 cycles of 5 seconds at 95°C, alternating with 20 seconds at 63°C, followed by a melt curve analysis. Relative mRNA quantities were determined by calibration of cycle threshold values to the standard curve of pooled complementary DNA samples. Results were normalized to cycle threshold values of cyclophilin. Iron levels were quantified using a chromagen reagent method.4Kohyama M. et al.Nature. 2009; 457: 318-321Crossref PubMed Scopus (314) Google Scholar Cellular viability was assessed using a CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) according to the manufacturer’s instructions. Whole-cell lysate and secretome samples underwent protein estimation using a Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). SILAC incorporates stable amino acid isotopes, without altering cellular biology, allowing direct comparison of the secretome by mass spectrometry between treatment groups.5Ong S.E. et al.Nat Protoc. 2006; 1: 2650-2660Crossref PubMed Scopus (684) Google Scholar SGBS pre-adipocytes were grown in SILAC Dulbecco's modified Eagle medium:F12 media (Thermo Fisher Scientific) supplemented with dialyzed fetal bovine serum (Thermo Fisher Scientific) and 22.81 mg/L 2H4-lysine and 36.88 mg/L 13C6-arginine (K4R6) or 22.81 mg/L 13C615N2-lysine and 36.88 mg/L 13C615N4-arginine (K8R10). Incorporation of labeled amino acids was confirmed by liquid chromatography tandem mass spectrometry on tryptic peptides prepared from whole-cell lysates. Cell pellets were lysed in 8 mol/L urea in 100 mmol/L triethylamonium bicarbonate, and protein concentration was estimated using the Bradford assay (BioRad, Hercules, CA). Thirty micrograms of cell lysate was reduced and alkylated by incubating samples for 30 minutes at 37°C with 2.5 mmol/L tris(2-carboxyethyl)phosphine and then 5 mmol/L 2-chloroacetamide. The urea concentration was diluted to 1 mol/L with 100 mmol/L triethylamonium bicarbonate before adding 0.6 μg of trypsin. Samples were incubated overnight then acidified to 1% trifluoroacetic acid and cleaned with OMIX C18 tips according to the manufacturers’ protocol (Agilent, Santa Clara, CA). Liquid chromatography tandem mass spectrometry was performed as described later. Labeled (>97%) cells underwent differentiation to adipocytes as described earlier. At day 14 after differentiation, cells were treated with vehicle (media) (K4R6, medium-weight cells) or 100 μmol/L FAC (in media) (K8R10, heavy-weight cells) for a further 48 hours using exactly equal volumes of media, replacing the media after 24 hours. Media for secretome analysis was collected from K4R6 and K8R10 cells and mixed 1:1 (vol/vol) before centrifugation at 600 × g at 4°C for 10 minutes to remove cell debris. Supernatant then was concentrated using Amicon Ultra 15 mL 10-kilodalton centrifugal filter units (Merck Millipore, Burlington, MA) in a fixed-angle centrifuge at 5000 × g to provide approximately 1-mL samples of concentrated mixed secretome. Thirty micrograms of protein was separated on 10% sodium dodecyl sulfate–polyacrylamide electrophoresis gels to 10 mm. Protein visualization, excision of bands, and in-gel trypsin digestion were performed using a semiautomated method as described.6Ruelcke J.E. et al.J Proteomics. 2016; 149: 3-6Crossref PubMed Scopus (19) Google Scholar A band corresponding to the same molecular weight as transferrin (media additive) was removed before digestion to provide a protein sample exclusively secreted from cultured adipocytes. A Q Exactive Plus Orbitrap Mass Spectrometer (Thermo Fisher Scientific), coupled with Easy-nLC 1000 and EASY-spray ion source (both Thermo Fisher Scientific), was used to analyze the digested peptides. Samples were loaded onto an EASY-Spray PepMap RSLC C18 2-μm column (50 cm × 75 μm ID), with a Nanoviper Acclaim C18 guard (75 μm × 2 cm) (both Thermo Fisher Scientific). A 90-minute method was run using a combination of buffer A (0.1% formic acid) and buffer B (0.1% formic acid:acetonitrile). A 2-step gradient was run comprising a 60-minute gradient from 3% to 25% buffer B and a 12-minute gradient from 25% to 40% buffer B. The flow rate was 250 nL/min. The mass spectrometer was programmed to acquire a full mass spectrometry resolution of 70,000 with an ACG target of 3 × 106, with a maximum injection time of 100 ms. The mass spectrometry scan range was from 350 to 1400 m/z. Tandem mass spectrometry was set to acquire a resolution of 17,500 with an ACG target of 5 × 105 and a maximum injection time of 55 ms. The loop count was set to 20 with a dynamic exclusion after 30 seconds. Raw data were processed with Spectrum mill (Rev B.05.00.181 SP1; Agilent). Selected modifications included fixed carbamidomethylation of cysteine and SILAC labels (Arg 0-6-10 daltons Lys 0-4-8 daltons) and variable oxidized methionine. Results were searched against the Human UniProt database (downloaded June 1, 2015).7The UniProt C. Nucleic Acids Res. 2017; : D158-D169PubMed Google Scholar Trypsin was selected as the digestion enzyme, with 2 maximum missed cleavages allowed. The precursor mass tolerance was set at ±20 ppm and product mass tolerance was ±20 ppm. Data were analyzed using the online Quantitative Proteomics P value Calculator using no normalization and nonadjusted P values.8Chen D. et al.J Proteome Res. 2014; 13: 4184-4191Crossref PubMed Scopus (22) Google Scholar Western blot using whole-cell lysate samples (10 μg) and 5-μL concentrated secretome samples was performed as previously described.9Britton L. et al.Physiol Rep. 2016; : e12837Crossref PubMed Scopus (3) Google Scholar A 1:500 dilution of primary antibody against ApoE (sc-53570; Santa Cruz, Dallas, TX) was applied to the membranes. A 1:100,000 dilution of goat anti-mouse horseradish peroxidase antibody (Invitrogen) was applied as secondary antibody. ApoE whole-cell lysate densitometry was normalized against densitometry using β-actin as a reference protein (1:2000 primary antibody) (cat no. 4967, Cell Signaling, Danvers, MA) and 1:20,000 goat anti-rabbit horseradish-peroxidase antibody (Invitrogen).Supplementary Figure 2Volcano plot of relative signal intensity of proteins identified in the adipocyte secretome. The x-axis denotes log2 of the ratio of iron/vehicle-treated cells, with proteins to the left of zero representing those down-regulated by iron and those to the right representing up-regulation by iron. The y-axis denotes statistical significance with a line representing a P value of .05. Proteins above this line have a P value <.05. SILAC-labeled adipocytes generated 338 proteins that were identified in the secretome by mass spectrometry. Of these, 213 had reduced signal intensity in response to iron, whereas 125 had increased signal intensity. Of the 213 proteins with reduced signal intensity, 61 had a statistically significant (P < .05) down-regulation in response to iron. Of these, 53 had a greater than 2-fold decrease in response to iron. Of the 125 proteins with increased signal intensity, 11 had a statistically significant (P < .05) up-regulation in response to iron. Of these, 7 proteins had a greater than 2-fold increase response to iron. Those proteins containing signal peptide (as determined by signal peptide annotations on the UniProt database) are shown in red. Those without signal peptide are shown in blue.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure 3Mechanistic aspects of iron-related dysregulation of protein secretion. (A) Interleukin 6 (IL6) mRNA (P = NS, paired t test, N = 3 per group). (B and C) Oxidative stress (*P < .05, both N = 3 per group). (B) Heme-oxygenase (HO-1) mRNA (P = .01, paired t test). (C) Glutathione peroxidase 1 (GPX1) mRNA (P = .049, paired t test). (D–I) Endoplasmic reticulum stress (all N = 3 per group). (D) Unspliced X-box binding protein (XBP1) mRNA (P = NS, paired t test). (E) Spliced XBP1 mRNA (P = NS, paired t test). (F) Immunoglobulin binding protein (BiP) mRNA (P = NS, paired t test). (G) Endoplasmic reticulum degradation-enhancing α-mannidose-like protein (EDEM) mRNA (P = NS, paired t test). (H) Activating transcription factor 4 (ATF4) mRNA (P = NS, paired t test). (I) CCAAT/enhancer-binding protein homologous protein (CHOP) mRNA (P = NS, paired t test). (J–M) Enrichment with signal peptide and exosome proteins. (J) Proportion of proteins down-regulated significantly by iron with signal peptide vs no signal peptide. (K) Proportion of proteins not down-regulated significantly by iron with signal peptide vs no signal peptide. (L) Proportion of proteins down-regulated significantly by iron with exosome secretion vs no exosome secretion. (M) Proportion of proteins not down-regulated significantly by iron with exosome secretion vs no exosome secretion. Of the 61 proteins down-regulated significantly, 62% (38 of 61) had signal peptide, whereas of the remaining proteins only 47% (129 of 277) had signal peptide. The 1-tailed Fisher exact test showed significant enrichment with signal peptide (P = .02) among the group down-regulated significantly. In contrast, there was no significant enrichment of the exosomal pathway (P = .51, 1-tailed Fisher exact test), because 15% (9 of 61) of the proteins down-regulated significantly and 14% (39 of 277) of the remaining secretome proteins had been reported previously in the high-confidence proteins from the EVpedia database.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Table 1List of SGBS Secretome Proteins With Significantly Altered Signal Intensity in Response to IronAccession numberGene nameProtein nameMean signal intensity ratio, iron/vehicleSDP valueQ8IX30SCUBE3Signal peptide, CUB and EGF-like domain-containing protein 30.0260.016.001P61353RPL2760S ribosomal protein L270.0600.005.001Q9NQH7XPNPEP3Probable Xaa-Pro aminopeptidase 30.0760.004.001P07996THBS1Thrombospondin-10.0830.047.001Q76M96CCDC80Coiled-coil domain-containing protein 800.0900.031.001P78539SRPXSushi repeat-containing protein SRPX0.0960.620.001Q9UHI8ADAMTS1A disintegrin and metalloproteinase with thrombospondin motifs 10.1140.090.002Q92538GBF1Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 10.1180.024.004Q15063POSTNPeriostin0.1210.149.001P08238HSP90AB1Heat shock protein (HSP) 90-β0.1250.038.004P24593IGFBP5Insulin-like growth factor-binding protein shock protein 75 and protein and a protein factor target A protein protein protein protein Proteins shown had a greater than 2-fold change in signal intensity in response to iron, with P < .05. Proteins in bold the 20 proteins of interest after of the UniProt protein Data were analyzed using the online Quantitative Proteomics P value Calculator using no normalization and nonadjusted P values = 3 per growth in a Proteins shown had a greater than 2-fold change in signal intensity in response to iron, with P < .05. Proteins in bold the 20 proteins of interest after of the UniProt protein Data were analyzed using the online Quantitative Proteomics P value Calculator using no normalization and nonadjusted P values = 3 per group). growth