MTBLS582: Global Analyses of Selective Insulin Resistance in Hepatocytes due to Palmitate Lipotoxicity

Abstract

Obesity is tightly linked to hepatic steatosis and insulin resistance. One feature of this association is the paradox of selective insulin resistance: insulin fails to suppress hepatic gluconeogenesis but activates lipid synthesis in the liver. How lipid accumulation interferes selectively with some branches of hepatic insulin signaling is not well understood. Here we provide a resource, based on unbiased approaches and established in a simple cell culture system, to enable investigations of the phenomenon of selective insulin resistance. We analyzed the phosphoproteome of insulin treated human hepatoma cells and identified sites in which palmitate selectively impairs insulin signaling. As an example, we show that palmitate interferes with insulin signaling to FoxO1, a key transcription factor regulating gluconeogenesis, and identify a possible mechanism. This model system, together with our comprehensive characterization of the proteome, phosphoproteome, and lipidome changes in response to palmitate treatment, provides a novel and useful resource for unraveling the mechanisms underlying selective insulin resistance.

Click to read more

 Authors: Tobias Walther, Robert Farese

  Release date: 26-Apr-2018

 Status: Public

Organism(s)

Homo sapiens

  Study Design

EFO:insulin resistance

CSEO:Lipidomics

EFO:Hepatic steatosis

GO:regulation of lipid biosynthetic process

untargeted metabolites

CHMO:ultra-performance liquid chromatography-mass spectrometry

CHMO:tandem mass spectrometry

  Experimental Factors

replicate

treatment

Protocol Description
Sample collection HepG2 cells were SILAC labeled and cultured in SILAC DMEM media with 10% (v/v) dialyzed FBS. For palmitate treatment, cells were seeded with 30% confluency, cultured for 18 h and treated with 0.25 mM palmitate for 24 h. Palmitate treated or untreated cells were stimulated with vehicle (50 µM HEPES, pH 8.0) or 100 nM insulin for 20 min and lysed in lysis buffer (6 M guanidinium hydrochloride (GdmCl), 10 mM Tris (2-carboxyethyl) phosphine (TCEP), 40 mM chloroacetamide, alkylating reagent (CAA), 100 mM Tris, pH 8.5).
Extraction The lipid extraction protocol was modified from a previously published procedure[1]. Briefly, cell lysates (biological triplicates – 200 µg of protein each, containing the following standard lipid mix (Avanti Polar Lipids): 375 pmoles C17:1 lysophosphatidic acid; 225 pmoles C17:0/C20:4 phosphatidic acid; 170 pmoles C17:1 lysophosphatidylserine; 180 pmoles C17:0/C20:4 phosphatidylserine; 86 pmoles C17:1 lysophosphatidylethanolamine; 112 pmoles C17:0/C14:1 phosphatidylethanolamine; 95 pmoles C17:1 lysophosphatidylcholine; 112 pmoles C17:0/C20:4 phosphatidylcholine; 33.2 pmoles C17:1 lysophosphatidylinositol; 165 pmoles C17:0/C20:4 phosphatidylinositol; 105 pmoles C17:0/C14:1 phosphatidylglycerol; 180 pmoles C17:0/d18:1 ceramide; 140 pmoles C17:0/d18:1 sphingomyelin; 155 pmoles C12:0/d18:1 monohexosyl-ceramide; and 60 pmoles C17:1/C17:1/C17:1 triacylglycerol) were suspended in ice-cold HPLC-grade water, and transferred to 13 x 100-mm Pyrex culture tubes with polytetrafluoroethylene (PTFE)-lined screw caps. Then, HPLC-grade water, methanol, and chloroform were added to each vial, generating a final chloroform/methanol/water (C/M/W) ratio of 1:2:0.8 (v/v/v). Samples were vortexed vigorously for 5 min, and centrifuged for 10 min at 1,800x g at room temperature. After centrifugation, the supernatants were collected and transferred to fresh vials, and the remaining pellets were dried under nitrogen stream. The dry pellets were extracted with chloroform/methanol (2:1, v/v), centrifuged, and the resulting supernatants were combined with the corresponding supernatants from the first step of extraction (C/M/W 1:2:0.8 v/v/v). The combined supernatants were dried under nitrogen stream, and the resulting samples were submitted to Folch’s partitioning by dissolving them in C/M/W (4:2:1.5, v/v/v), followed by vortexing and centrifuging, as described above. After centrifugation, the lower (organic) and upper (aqueous) phases were separated, and transferred to fresh Pyrex glass test tubes. The Folch upper phase was then re-extracted with C/M (2/1; v/v), and the resulting organic phase was combined with the organic phase from the preceding step. The pooled organic phases were dried under a N2 stream, and stored at -20 °C until use.

Ref:
[1] Gazos-Lopes, F., Oliveira, M. M., Hoelz, L. V., Vieira, D. P., Marques, A. F., Nakayasu, E. S., Gomes, M. T., Salloum, N. G., Pascutti, P. G., Souto-Padron, T., Monteiro, R. Q., Lopes, A.H., and Almeida, I. C. (2014) Structural and functional analysis of a platelet-activating lysophosphatidylcholine of Trypanosoma cruzi. PLoS Negl Trop Dis 8, e3077. doi:10.1371/journal.pntd.0003077. PMID:25101628
Chromatography Lipid samples extracted as described above were diluted in 50 µl of C/M (2:1; v/v) and analyzed by UHPLC-ESI-MS using a modification of the method previously described[1]. Briefly, UHPLC-ESI-MS/MS was conducted using a Dionex™ UltiMate 3000™ UHPLC system (Thermo Scientific) coupled to Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific). 5 µl injections of samples were made onto an Accucore™ (Thermo Scientific) C18 LC column (2.6 µm solid-core particles, length of 30 mm). The eluent A consisted of acetonitrile/water (50:50 v/v) + 10 mM ammonium formate and 0.2% formic acid, and the eluent B consisted of methanol/isopropanol/water (10:88:20 v/v/v) + 2 mM ammonium formate and 0.01 % formic acid. The following program was used for gradient elution of analytes (total run time of 46 min) at a constant flow rate of 500 ml/min. 35% eluent B – 45% eluent B over 5 min, 45% eluent B – 85% eluent B from min 5 – min 28, 85% eluent B – 100% eluent B from min 28 – min 38, followed by an immediate drop back to 35% eluent B, which is held constant up to min 46 of each run. The column temperature was set to 35 °C, and the autosampler tray temperature was set to 10 °C.

Ref:
[1] Fröhlich F, Petit C, Kory N, Christiano R, Hannibal-Bach HK, Graham M4, Liu X, Ejsing CS, Farese RV, Walther TC. The GARP complex is required for cellular sphingolipid homeostasis. Elife. 2015 Sep 10;4. doi:10.7554/eLife.08712. PMID:26357016
Mass spectrometry The ion source was a HESI II (Thermo Scientific) set in the following conditions: sheath gas flow rate set to 60; auxiliary flow rate set to 20; sweep gas flow rate set to 1; spray voltage (KV) set to 3.00; capillary temperature set to 285 °C, S-Lens RF level set to 45, and auxiliary temperature set to 370 °C. Samples were run in either positive or in negative ion modes with the mass spectrometer acquisition settings set as follows. For Full Scan – top 15 data-dependent MS/MS: scan range of 250-1800 m/z; mass resolution was set to 70,000; AGC target was set to 1e^6; the C-trap ion accumulation time was set to 120 ms. For Data-dependent MS/MS: mass resolution was set to 30,000; AGC target was set to 5e^5; the C-trap ion accumulation time was set to 120 ms; select ion exclusion was set to 8 s; and the HCD (higher-energy collisional dissociation) fragmentation ramp was set to 15, 25, and 35 NCE (normalized collision energy) respectively.
Data transformation No data transformation was required
Metabolite identification Raw files generated by the LC-MS/MS runs were analyzed using the LipidSearchTM Software (Thermo Scientific) for the identification of lipid species, combining accurate-mass information from parent ion with MS2 information. The following parameters were used for batch analysis: Database – Q Exactive; Search Type – Product; Experiment Type (Exp Type) - LC-MS; Parent Tolerance (Parent Tol) - 0.1 Da; NL/Prec Tol – 0.5 Da; Precursor Tolerance (Prec Tol) - 10.0 ppm; Product Tolerance (15.0) ppm; Merge Range (Min) - 0.0; Minimal Peak Width (min) - 0.0; Threshold Type - Relative; Product Ion – 1.0%; m-score threshold – 2.0; Recalculate Isotope – ON; R.T. Interval (min) - 0.01; Execute Quantitation – ON; m/z Tolerance (m/z tol) - -10.0/+10.0; Tolerance Type – ppm; R.T. range (min) - -0.05/+0.05; Toprank filter – ON; Main Node Filter – Main Isomer Peak; m-Score Threshold (Display) - 5.0; C-Score Threshold (Display) - 2.0; Fatty Acid Priority (FA Priority) - ON; ID Quality Filter (A; B; C). The following parameters were used for sample alignment: Search Type – Product; Experiment Type (ExpType) - LC–MS; Alignment Method – Max; R.T. Tolerance – 0.25; Calculate Unassigned Peak Area – ON; Filter Type – New Filter; Toprank Filter –ON; Main Node Filter – Main Isomer Peak; m-score threshold – 5.0; ID Quality – A,B,C. Because this study required a careful comparison between the lipid composition of T. cruzi and its host cells, all lipid species identified using the LipidSearchTM software were manually curated after computational analysis, being either accepted, rejected, or reassigned.

Known quantities of each lipid standard were analyzed using the same UHPLC-ESIMS/MS methods to generate a response factor (RF; peak area/pmol standard injected). The RF for each standard was divided by the CerG1 RF to calculate a molar relative response factor (MRRF) for each major lipid class. The MRRF for each class was normalized to the CerG1 peak area of each sample, and peak areas adjusted accordingly. Only those classes for which standards were detected in each run were considered for analysis. The in-built statistical tool for the quantitation of lipid species LipidSearchTM is student t-test. For the calculation of lipid class fatty acid composition, the MainArea values output by LipidSearch™ were assigned to each FA moiety of a given species and summed using Microsoft Excel PivotTable. Area % is the summed FA value divided by the lipid class total, multiplied by 100 to reflect percentage within each lipid class.
Source Name Organism Organism part Protocol REF Sample Name replicate treatment
WT_ZL_01_Neg Homo sapiens Hep-G2 cell Sample collection WT_ZL_01_Neg 1 control
WT_ZL_01_Pos Homo sapiens Hep-G2 cell Sample collection WT_ZL_01_Pos 1 control
WT_ZL_02_Neg Homo sapiens Hep-G2 cell Sample collection WT_ZL_02_Neg 2 control
WT_ZL_02_Pos Homo sapiens Hep-G2 cell Sample collection WT_ZL_02_Pos 2 control
WT_ZL_03_Neg Homo sapiens Hep-G2 cell Sample collection WT_ZL_03_Neg 3 control
WT_ZL_03_Pos Homo sapiens Hep-G2 cell Sample collection WT_ZL_03_Pos 3 control
Palm_ZL_01_Neg Homo sapiens Hep-G2 cell Sample collection Palm_ZL_01_Neg 1 palmitate
Palm_ZL_01_Pos Homo sapiens Hep-G2 cell Sample collection Palm_ZL_01_Pos 1 palmitate
Palm_ZL_02_Neg Homo sapiens Hep-G2 cell Sample collection Palm_ZL_02_Neg 2 palmitate
Palm_ZL_02_Pos Homo sapiens Hep-G2 cell Sample collection Palm_ZL_02_Pos 2 palmitate
Palm_ZL_03_Neg Homo sapiens Hep-G2 cell Sample collection Palm_ZL_03_Neg 3 palmitate
Palm_ZL_03_Pos Homo sapiens Hep-G2 cell Sample collection Palm_ZL_03_Pos 3 palmitate

Assay  1

Assay file name: a_mtbls582_NEG_mass_spectrometry.txt
Technology: mass spectrometry
Platform: Exactive (Thermo Scientific)

Instrumentation

Sample Name Protocol REF Post Extraction Derivatization Extract Name Protocol REF Chromatography Instrument Column model Column type Labeled Extract Name Label Protocol REF Scan polarity Scan m/z range Instrument Ion source Mass analyzer MS Assay Name Raw Spectral Data File Protocol REF Normalization Name Derived Spectral Data File Protocol REF Data Transformation Name Metabolite Assignment File
WT_ZL_01_Neg Extraction 50 µl chloroform/methanol, 2/1 (v/v) none Chromatography Thermo Scientific Dionex Ultimate 3000 UHPLC system Accucore C18 (2.6 µm, 2.1 mm x 30 mm; Thermo Scientific) reverse phase Mass spectrometry negative 250-1800 Thermo Scientific Q Exactive electrospray ionization orbitrap WT_ZL_01_Neg WT_ZL_01_Neg.raw Data transformation Metabolite identification m_mtbls582_NEG_mass_spectrometry_v2_maf.tsv
WT_ZL_02_Neg Extraction 50 µl chloroform/methanol, 2/1 (v/v) none Chromatography Thermo Scientific Dionex Ultimate 3000 UHPLC system Accucore C18 (2.6 µm, 2.1 mm x 30 mm; Thermo Scientific) reverse phase Mass spectrometry negative 250-1800 Thermo Scientific Q Exactive electrospray ionization orbitrap WT_ZL_02_Neg WT_ZL_02_Neg.raw Data transformation Metabolite identification m_mtbls582_NEG_mass_spectrometry_v2_maf.tsv
WT_ZL_03_Neg Extraction 50 µl chloroform/methanol, 2/1 (v/v) none Chromatography Thermo Scientific Dionex Ultimate 3000 UHPLC system Accucore C18 (2.6 µm, 2.1 mm x 30 mm; Thermo Scientific) reverse phase Mass spectrometry negative 250-1800 Thermo Scientific Q Exactive electrospray ionization orbitrap WT_ZL_03_Neg WT_ZL_03_Neg.raw Data transformation Metabolite identification m_mtbls582_NEG_mass_spectrometry_v2_maf.tsv
Palm_ZL_01_Neg Extraction 50 µl chloroform/methanol, 2/1 (v/v) none Chromatography Thermo Scientific Dionex Ultimate 3000 UHPLC system Accucore C18 (2.6 µm, 2.1 mm x 30 mm; Thermo Scientific) reverse phase Mass spectrometry negative 250-1800 Thermo Scientific Q Exactive electrospray ionization orbitrap Palm_ZL_01_Neg Palm_ZL_01_Neg.raw Data transformation Metabolite identification m_mtbls582_NEG_mass_spectrometry_v2_maf.tsv
Palm_ZL_02_Neg Extraction 50 µl chloroform/methanol, 2/1 (v/v) none Chromatography Thermo Scientific Dionex Ultimate 3000 UHPLC system Accucore C18 (2.6 µm, 2.1 mm x 30 mm; Thermo Scientific) reverse phase Mass spectrometry negative 250-1800 Thermo Scientific Q Exactive electrospray ionization orbitrap Palm_ZL_02_Neg Palm_ZL_02_Neg.raw Data transformation Metabolite identification m_mtbls582_NEG_mass_spectrometry_v2_maf.tsv
Palm_ZL_03_Neg Extraction 50 µl chloroform/methanol, 2/1 (v/v) none Chromatography Thermo Scientific Dionex Ultimate 3000 UHPLC system Accucore C18 (2.6 µm, 2.1 mm x 30 mm; Thermo Scientific) reverse phase Mass spectrometry negative 250-1800 Thermo Scientific Q Exactive electrospray ionization orbitrap Palm_ZL_03_Neg Palm_ZL_03_Neg.raw Data transformation Metabolite identification m_mtbls582_NEG_mass_spectrometry_v2_maf.tsv

Assay  2

Assay file name: a_mtbls582_POS_mass_spectrometry.txt
Technology: mass spectrometry
Platform: Exactive (Thermo Scientific)

Instrumentation

Sample Name Protocol REF Post Extraction Derivatization Extract Name Protocol REF Chromatography Instrument Column model Column type Labeled Extract Name Label Protocol REF Scan polarity Scan m/z range Instrument Ion source Mass analyzer MS Assay Name Raw Spectral Data File Protocol REF Normalization Name Derived Spectral Data File Protocol REF Data Transformation Name Metabolite Assignment File
WT_ZL_01_Pos Extraction 50 µl chloroform/methanol, 2/1 (v/v) none Chromatography Thermo Scientific Dionex Ultimate 3000 UHPLC system Accucore C18 (2.6 µm, 2.1 mm x 30 mm; Thermo Scientific) reverse phase Mass spectrometry positive 250-1800 Thermo Scientific Q Exactive electrospray ionization orbitrap WT_ZL_01_Pos WT_ZL_01_Pos.raw Data transformation Metabolite identification m_mtbls582_POS_mass_spectrometry_v2_maf.tsv
WT_ZL_02_Pos Extraction 50 µl chloroform/methanol, 2/1 (v/v) none Chromatography Thermo Scientific Dionex Ultimate 3000 UHPLC system Accucore C18 (2.6 µm, 2.1 mm x 30 mm; Thermo Scientific) reverse phase Mass spectrometry positive 250-1800 Thermo Scientific Q Exactive electrospray ionization orbitrap WT_ZL_02_Pos WT_ZL_02_Pos.raw Data transformation Metabolite identification m_mtbls582_POS_mass_spectrometry_v2_maf.tsv
WT_ZL_03_Pos Extraction 50 µl chloroform/methanol, 2/1 (v/v) none Chromatography Thermo Scientific Dionex Ultimate 3000 UHPLC system Accucore C18 (2.6 µm, 2.1 mm x 30 mm; Thermo Scientific) reverse phase Mass spectrometry positive 250-1800 Thermo Scientific Q Exactive electrospray ionization orbitrap WT_ZL_03_Pos WT_ZL_03_Pos.raw Data transformation Metabolite identification m_mtbls582_POS_mass_spectrometry_v2_maf.tsv
Palm_ZL_01_Pos Extraction 50 µl chloroform/methanol, 2/1 (v/v) none Chromatography Thermo Scientific Dionex Ultimate 3000 UHPLC system Accucore C18 (2.6 µm, 2.1 mm x 30 mm; Thermo Scientific) reverse phase Mass spectrometry positive 250-1800 Thermo Scientific Q Exactive electrospray ionization orbitrap Palm_ZL_01_Pos Palm_ZL_01_Pos.raw Data transformation Metabolite identification m_mtbls582_POS_mass_spectrometry_v2_maf.tsv
Palm_ZL_02_Pos Extraction 50 µl chloroform/methanol, 2/1 (v/v) none Chromatography Thermo Scientific Dionex Ultimate 3000 UHPLC system Accucore C18 (2.6 µm, 2.1 mm x 30 mm; Thermo Scientific) reverse phase Mass spectrometry positive 250-1800 Thermo Scientific Q Exactive electrospray ionization orbitrap Palm_ZL_02_Pos Palm_ZL_02_Pos.raw Data transformation Metabolite identification m_mtbls582_POS_mass_spectrometry_v2_maf.tsv
Palm_ZL_03_Pos Extraction 50 µl chloroform/methanol, 2/1 (v/v) none Chromatography Thermo Scientific Dionex Ultimate 3000 UHPLC system Accucore C18 (2.6 µm, 2.1 mm x 30 mm; Thermo Scientific) reverse phase Mass spectrometry positive 250-1800 Thermo Scientific Q Exactive electrospray ionization orbitrap Palm_ZL_03_Pos Palm_ZL_03_Pos.raw Data transformation Metabolite identification m_mtbls582_POS_mass_spectrometry_v2_maf.tsv
   Download study (FTP)  |     Download metadata    

Aspera Download Details:

List of study files   Subset

File
Palm_ZL_01_Pos.raw
Palm_ZL_01_Neg.raw
Palm_ZL_03_Neg.raw
Palm_ZL_03_Pos.raw
WT_ZL_01_Pos.raw
Palm_ZL_02_Neg.raw
WT_ZL_03_Neg.raw
WT_ZL_02_Neg.raw
WT_ZL_02_Pos.raw
audit
WT_ZL_01_Neg.raw
WT_ZL_03_Pos.raw
Palm_ZL_02_Pos.raw
metexplore_mapping.json
a_mtbls582_NEG_mass_spectrometry.txt
a_mtbls582_POS_mass_spectrometry.txt
i_Investigation.txt
MTBLS582_T1.xls
m_mtbls582_NEG_mass_spectrometry_v2_maf.tsv
m_mtbls582_POS_mass_spectrometry_v2_maf.tsv
s_mtbls582.txt

Info:To download a single file, just click on the file name. To download multiple files, use the check boxes and then click "Download selected files" button. Files will be zipped and downloaded to your browser.



Validations marked with (*) have been allowed by the MetaboLights Curators.
Click here for the detailed description of Validations.
Condition Status Description Requirement Group Message
PASSES Study Title MANDATORY STUDY OK
PASSES Study Description MANDATORY STUDY OK
PASSES Study text successfully parsed OPTIONAL STUDY OK
PASSES Study Contact(s) have listed email MANDATORY CONTACT OK
PASSES Sample(s) MANDATORY SAMPLES OK
PASSES Sample Name consistency check MANDATORY ASSAYS OK
PASSES Publication(s) associated with this Study MANDATORY PUBLICATION OK
PASSES Minimal Experimental protocol MANDATORY PROTOCOLS OK
PASSES Comprehensive Experimental protocol OPTIONAL PROTOCOLS OK
PASSES Extraction protocol description MANDATORY PROTOCOLS OK
PASSES Data transformation protocol description MANDATORY PROTOCOLS OK
PASSES Metabolite Identification protocol description MANDATORY PROTOCOLS OK
PASSES Mass spectrometry protocol description MANDATORY PROTOCOLS OK
PASSES Chromatography protocol description MANDATORY PROTOCOLS OK
PASSES Sample Collection protocol description MANDATORY PROTOCOLS OK
PASSES Protocols text successfully parsed OPTIONAL PROTOCOLS OK
PASSES Organism name MANDATORY ORGANISM OK
PASSES Organism part MANDATORY ORGANISM OK
PASSES Study Factors MANDATORY FACTORS OK
PASSES Assay platform information OPTIONAL ASSAYS OK
PASSES Assay has raw files referenced MANDATORY FILES OK
PASSES Assay referenced raw files detection in filesystem MANDATORY FILES OK
PASSES Raw files in the Assay(s) have the correct format MANDATORY FILES OK
PASSES Assay(s) MANDATORY ASSAYS OK
PASSES All Assays have Metabolite Assignment File (MAF) referenced OPTIONAL FILES OK
PASSES Metabolite Assignment File (MAF) is present in Study folder MANDATORY FILES OK
PASSES Metabolite Assignment File (MAF) has correct format MANDATORY FILES OK
PASSES Metabolite Identification File (MAF) content MANDATORY FILES OK
PASSES ISA-Tab investigation file check MANDATORY ISATAB OK

Pathways - Assay  1



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)

Pathways - Assay  2



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)