AML 12 cells were purchased from American Type Culture Collection (ATCC, RRID: CVCL_0140), and they were tested negative for mycoplasma contamination. AML12 were cultured in 1:1 (v/v) Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (Thermo), supplemented with 10% foetal bovine serum (FBS), 1% penicillin/streptomycin (100 units/mL and 100 μg/mL, respectively), 1% insulin-transferrin-selenium (ITS; 10 mg/L, 5.5 mg/L and 6.7 μg/L respectively), and dexamethasone (100 μmol/L) at 37 °C in 5% CO2. Cells were plated at a density of 50,000 cells/well in collagen 1-coated 12-well plates (Cat. # 7340295, Corning) and grown to confluence in maintenance medium.
Naa40, Acss2, or NAA40 + Accs2 in AML 12 cells were silenced by siRNA transfection (Horizon Discovery). In brief, cells were plated at a density of 100,000 cells/well and on day 2, cells were transfected with 25 nM siRNA specific for Naa40 (QIAgen; cat. # GS70999) or Acss2 (QIAgen; cat. # GS60525) or negative control (QIAgen; cat. #1027281) or water (mock) using DharmaFECT 1 Transfection Reagent (Horizon discovery) according to the manufacturer’s instructions. Cells were harvested at 6, 12, 24, and 48 h after transfection.
Transfected cells were treated in presence or absence of insulin (10 mg/L; Gibco cat. #41400045) or the insulin analogue insulin lispro (10 mg/L; Sigma, cat. #1342321).
Transfected cells were treated with 5 mM sodium butyrate (Sigma, Cat. B5887), for 48 h.
Lipid synthesis inhibition
Transfected cells were treated with 30 μM FSG67, a GPAT inhibitor (Focus Biomolecule, Cat. 10-4577), for 48 h.
Transfected cells were treated with 1 μg/ml of Actinomycin D, a class II gene transcription inhibitor (Santa Cruz, Cat. # CAS 50-76-0.), for 24 h.
Metabolites and lipids were extracted from cells using a modified method of Folch and colleagues . Briefly, pelleted cells were homogenised in chloroform/methanol (2:1, v/v, 750 μL); including a mixture of deuterated internal standards. Samples were sonicated for 15 min and deionised water was added (300 μL). The organic (upper layer) and aqueous (lower layer) phases were separated following centrifugation at 13,000×g for 20 min. The organic phase extracts containing lipids were dried under a stream of nitrogen gas whilst the aqueous samples were dried in a CentriVap Centrifugal Concentrator with attached cold trap (78100 series, Labconco Co, Kansas City, USA).
Analysis of aqueous metabolites by triple quadrupole mass spectrometry
Aqueous extracts were reconstituted in acetonitrile: 10 mM ammonium carbonate (7:3, v/v, 50 μL) containing an internal standard mix (AMP 13C10, 15N5; ATP 13C10, 15N5; Glutamate U13C, U15N; Leucine-d10, Phenylalanine-d5, Proline U13C, U15N; and Valine-d8). Samples were injected onto a Vanquish UHPLC attached to a TSQ Quantiva triple quadrupole mass spectrometer (Thermo Scientific) with a heated ESI source. Samples were analysed with normal phase and reverse phase chromatography.
For the normal phase analysis, metabolites were separated with a BEH-amide (150 × 2.1 mm 1.7 μm) column at 30 °C. The mobile phase consisted of (A) 0.1% of ammonium carbonate and (B) acetonitrile and was pumped at a flow rate of 0.6 mL/min. The gradient was programmed as follows: 80% of B for 1.50 min followed by a linear decrease from 80 to 40% of B for 3.5 min and finally returned to initial conditions.
For reverse phase analysis, samples were dried and reconstituted in 10 mM ammonium acetate solution and analysed with an ACE C18 PFP (150 × 2.1 mm 5 μm) column at 30 °C. The mobile phase consisted of (A) 0.1 % formic acid in water and (B) 0.1 % formic acid in acetonitrile, pumped at 0.5 mL/min. The gradient was programmed as follows: 0% of B for 1.60 min followed by a linear increase from 0 to 30% of B for 4 min and to 90% by 4.5 min, held for 1 min and then returned to initial conditions.
The mass spectrometer was operated in SRM mode; collision energies and RF lens voltages were generated for each species using the TSQ Quantiva optimisation function. Xcalibur Software (Thermo Scientific) was used to identify peaks, process mass spectra, and normalise data to the closest-eluting internal standard.
Analysis of intact lipids using high-resolution mass spectrometry
To the organic fraction an internal standard mix was incorporated [N-palmitoyl-d31-D-erythro-sphingosine (16:0-d31 Ceramide), pentadecanoic-d29 acid (15:0-d29 FFA), heptadecanoic-d33 acid (17:0-d33 FFA), eicosanoic-d39 acid (20:0-d39 FFA), 1-palmitoyl(D31)-2-oleyl-sn-glycero-3-phosphatidylcholine (16:0-d31-18:1 PC), 1-palmitoyl(d31)-2-oleyl-sn-glycero-3-phosphoethanolamine (16:0-d31-18:1 PE), 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (16:0-d31-18:1 PG), N-palmitoyl(d31)-d-erythro-sphingosylphosphorylcholine (16:0-d31 SM), glyceryl tri(pentadecanoate-d29) (45:0-d87 TAG), glyceryl tri(hexadecanoate-d31) (48:0-d93 TAG) (Avanti Polar Lipids Inc, USA)] and dried down under nitrogen. The organic fraction was reconstituted in 100 μL chloroform/methanol (1:1, v/v), and 10 μL of the resulting solution added to 90 μL isopropanol (IPA)/acetonitrile (ACN)/water (2:1:1, v/v). Analysis of the fractions was performed using an LTQ Orbitrap Elite Mass Spectrometer (Thermo Scientific, Hemel Hempstead, UK).
In positive mode, 5 μL of sample were injected onto a C18 CSH column, 1.7 μM pore size, 2.1 mm × 50 mm (Cat # 186005296, Waters Ltd, Manchester, UK) which was held at 55 °C in a Dionex Ultimate 3000 ultra-high performance liquid chromatography system (UHPLC; Thermo Scientific). A gradient (flow rate 0.5 mL/min) of mobile phase A (ACN/water 60:40, 10 mmol/L ammonium formate) and B (LC–MS-grade ACN/IPA 10:90, 10 mmol/L ammonium formate) was used. In negative ion mode, 10 μL of the sample was injected and 10 mmol/L ammonium acetate was used as the additive to aid ionisation.
In both positive and negative ion mode, the gradient began at 40% B, increased to 43% B at 0.8 min, 50% B at 0.9 min, 54% B at 4.8 min, 70% B at 4.9 min, 81% B at 5.8 min, peaked at 99% B at 8 min for 0.5 min, and subsequently returned to the starting conditions for another 1.5 min to re-equilibrate the column. The UHPLC was coupled to an electrospray ionisation (ESI) source which ionised the analytes before entering the mass spectrometer. Data were collected in both positive and negative ion mode with a mass range of 110–2000 m/z. Default instrument-generated optimisation parameters were used.
The spectra files were converted to mzML format and features picked using xcms , after retention time alignment. Lipid identification was performed using an in-house R script. Peak areas of each metabolite were normalised to the appropriate internal standard and tissue weight.
Glucose assay uptake
A 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) glucose uptake assay was performed according to the manufacturer’s instructions (Biovision Milpitas, CA). In brief, after starving cells for 6 h with 0.5% FBS-containing DMEM, 2-NBDG was added for 30 min at 37 °C in 5% CO2. Cells were harvested with trypsin and centrifugation at 1200 rpm for 5 min. The pellet was resuspended in PBS and the fluorescent uptake was measured using flow cytometry. Data were acquired on a Bio-Rad S3e Cell Sorter and analysed using FlowJo software (Treestar).
Fly stocks and crosses
Fly stocks used in this study are w1118 (BDSC# 3605, RRID: BDSC_3605); 0.68-lsp2 Gal4 [kindly provided from Brigitte Dauwalder, University of Houston]  and UAS-Naa40RNAi (VDRC# KK101213) . Transgenic RNAi fly stocks were obtained from the Vienna Drosophila Resource Center (VDRC, www.vdrc.at). For FB-specific Naa40 KD, 0.68-lsp2 GAL4 virgin females were crossed to UAS-Naa40RNAi males, whereas, for the control conditions, GAL4 virgin females were crossed to w1118 males. All flies and crosses were maintained in 25 °C under a 12-h light/dark circle and crosses were allowed to proceed only for 1–2 days to avoid larval crowding. For our studies, we chose to use only male L3 progeny except the TG analysis were both male and female larvae were used.
Gene expression analysis
Total RNA was extracted and purified from hepatocytes using a RNeasy Mini Kit (QIAgen) according to the manufacturer’s specifications. Purified RNA concentration was quantified at 260 nm using a NanoDrop 100 (Thermo Fisher Scientific). Third instar larval fat body total RNA (8-10 fat bodies per sample) was extracted using Trizol via guanidinium thiocyanate-phenol-chloroform using TRIzol™ Reagent (Thermo Fischer Scientific).
Each purified RNA sample was diluted with RNase-free water to a final concentration of 100 ng/μL. Complementary DNA (cDNA) synthesis and genomic DNA elimination in RNA samples was performed using an RT2 First Strand Synthesis kit (QIAgen) according to the manufacturer’s specifications. The reactions were stored at − 20 °C prior to real-time quantitative PCR analysis. The relative abundance of transcripts of interest was measured by qPCR in KAPA SYBR Green (SYBR Green Fast qPCR Master Mix) with a the Bio-Rad CFX96 detection system (Applied Biosystems). The SYBR Green qPCR Mastermix contained HotStart DNA Taq Polymerase, PCR Buffer, dNTP mix (dATP, dCTP, dGTP, dTTP), and SYBR Green dye. Before adding cDNA to each well of the 96-well plate, cDNA was diluted in RNase-free water to a final concentration of 8 ng/μL. PCR component mix was prepared by mixing 10 μL SYBR Green qPCR Mastermix with 0.6 μL of 10 μmol/L target primers (forward and reverse; 6 pmoles/reaction) and 4.4 μL RNase-free water. To each well of a 96-well plate, 5 μL cDNA (total amount 40 ng) and 15 μL PCR components mix were added. The plate was centrifuged at 1000×g for 30 s to ensure that the contents were mixed and to remove any bubbles present in the wells. The plate was placed in the real-time cycler with the following cycling conditions: 10 min at 95 °C for 1 cycle to activate HotStart DNA Taq Polymerase; 15 s at 95 °C and 1 min at 60 °C to perform elongation and cooling for 40 cycles. Sequences for qPCR Primers used for mouse Rn18s, Fasn, Acaca, Gpat1, Agpat1, Pap, Dgat1, Hsl, and Atgl and Drosophila melanogaster actin, naa40, fasn and lipin were purchased from Integrated DNA Technologies (Additional file 7: Table S2). Expression levels were normalised to the endogenous control, Rn18s for mouse or actin for drosophila, using the ΔΔCt method and fold changes reported were relative to the control group in the dose response (mock).
Histones were purified using the method of Shechter et al. .
Sample processing for triglyceride quantification was performed as described in Werthebach et al. (2019) . Briefly, three to six crawling L3 larvae were transferred in a 1.5-mL Eppendorf tube and homogenised in 100 μL 0.05% Tween20 in H2O per larva, using a motorised homogeniser. Then, the homogenate was heat inactivated by incubation at 70 °C for 5 min and centrifuged at 5000 rpm for 5 min. The supernatant was collected in a new tube and centrifuged at 14,800 rpm for 15 min at 4 °C. Triglyceride measurement was performed using the Triglyceride (TG) Colorimetric Assay Kit (Elabscience, USA) according to the manufacturer’s instructions. Protein values used to normalise TG measurements were quantified using the Bradford method. Both male and female larvae samples were collected and following comparison of TG levels with no sex-specific changes found, male and female measurements were pooled.
Cell pellets and Drosophila larvae (3–5 whole third instar larvae/replicate) were lysed in 100 μL Cell Extraction Buffer (10 mmol/L Tris, 100 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 10 mmol/L NaF, 20 mmol/L Na4P2O7, 20 mmol/L Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulfate, 0.5% deoxycholate, 1 mmol/L phenylmethylsulfonyl fluoride, complete protease inhibitor tablet, and 1% of each phosphatase cocktail inhibitor 2 and 3) for 30 min, vortexing at 10-min intervals. The lysate was centrifuged at 13,000×g for 10 min at 4 °C and the supernatant was collected and stored at − 80 °C.
The protein concentration was measured by Bradford assay (Bio-Rad). Approximately 20–50 μg of protein was separated on SDS-PAGE and subsequently transferred to a nitrocellulose membrane (GE Healthcare). The membranes were blocked with 5% TBS-T/BSA for 1 h at room temperature and then incubated with the primary antibodies at 4 °C overnight. The primary antibodies used were as follows: NAA40 (1:1000, ab106408, Abcam, RRID: AB_10866699), H2AS1Ph/ H4S1PHh (1:1000, ab177309, Abcam, RRID: AB_2797594), phospho-Akt (Ser473) (1:1000, cat. 9271, Cell signalling, RRID: AB_329825), Akt (pan) (1:000, cat. C67E7, Cell Signaling, RRID: AB_915783), H4K5ac (0.5:25,000; cat. ab51997, Abcam, RRID: AB_2264109), H4K8ac (1:1000; cat. ab15823, Abcam, RRID: AB_880455), H4K12ac (1:5000; cat. A b46983, Abcam, RRID: AB_873859), H4K16ac (1:1000; cat. ab109463, Abcam, RRID: AB_10858987), H3K64ac (1:1000; cat. ab214808, Abcam, RRID: AB_2894996), H4 (1:1000; cat. 05-858, Millipore, RRID: AB_390138), H3 (1:000, ab1791, Abcam, RRID: AB_302613) and β-actin (1:1000; sc-1616-R, Santa Cruz, RRID: AB_630836). The membranes were incubated with the secondary antibody, horseradish peroxide (HRP)-conjugated goat anti-rabbit IgG (1:30,000, Scientific). Bands were visualised by the enhanced chemiluminescence system (BioRad) and analysed using densitometry using ImageJ analysis software (NJH).
Original immunoblot images can be found in Additional file 8: Fig S6.
Lipid staining in AML12
Cells were seeded on coverslips in 12-well plates. Cells were washed with 1× PBS and fixed with 4% paraformaldehyde. Intracellular lipids were stained with Nile red (Invitrogen) as described by the manufacturer. Nuclei/DNA were stained with DAPI (Dako). Images were obtained using a Zeiss Axiovert 200 M microscope.
Nile red (NR) staining of larval fat body and imaging
Fat bodies (FBs) from crawling third instar (L3) larvae were dissected in PBS and then fixed in 4% formaldehyde in PBS, for 30 min at room temperature. Following PBS rinses, FBs were incubated for 1 h in NR solution, in the dark. Then, excess NR was rinsed with water and samples were mounted on microscopy slides for imaging using 75% glycerol. Samples were imaged using Leica TCS SP2 AOBS DMIRE2 inverted scanning confocal microscope and images were visualised using ImageJ.
FB thickness was calculated by addition of the number of optical sections required to complete a full z-stack, with the top and bottom sections representing the immediately following section before no sample was visible. The number of LDs per fat cell were quantified in Adobe Photoshop CC 2017 with the count tool, using a modified version of Fan et al. . Specifically, the number of LDs in 10 fat cells, from at least 3 biological samples were counted.
FBs were fixed in 4% formaldehyde for 20 min at room temperature and then washed with 0.2% PBS in Triton (0.2% PBSTr). Following 1 h blocking with 10% heat inactivated goat serum in 0.2% PBSTr, FBs were incubated with Alexa 488 Phalloidin (Molecular Probes, 1:500) in block for 2 h at room temperature. Following a brief rinse, samples were incubated with DAPI (1 μg/ul) for 10 min at room temperature. Then samples were washed with PBS and mounted on microscopy slides with Vectashield and imaged on a Zeiss Axioscope A.1 fluorescent microscope.
Multivariate statistical analyses were performed in Metaboanalyst 4.0 (www.metaboanalyst.ca). All variables were log transformed and subjected to principal component analysis (PCA) and pathway analysis and metabolite set enrichment analysis of significant metabolites. The extent to which the model fits and predicts the data is represented by R2 X and Q2 X, respectively.
Data were visualised using GraphPad (GraphPad Prism 8.0; GraphPad Software, San Diego, CA, USA). All data are expressed as means ± SEM. In GraphPad, unpaired t-test, one- or two-way ANOVA was performed where appropriate to determine significant differences between experimental groups. For a single comparison between two groups, an unpaired 2-tailed t-test was applied. For a single comparison between more than two groups, one-way ANOVA was used, whilst for two comparisons between more than two groups two-way ANOVA was used. For one-way ANOVA, Tukey’s post hoc multiple comparison test was performed, whilst for two-way ANOVA, Sidak’s post hoc multiple comparison test was used. Differences between experimental groups were statistically significant when p ≤ 0.05. All experiments were reproduced at least three times.