Home Nature Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes

Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes

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Mice

The next mouse strains have been used: Dars2fl/fl (ref. 2), Cox10fl/fl (ref. 4), Vil1-cre (ref. 7) and Villin-creERT2 (ref. 37). Sdhatm2a mice have been obtained from the Knock Out Mouse Mission repository (challenge ID: CSD48939) and bred to FLP deleter mice38 to delete the FRT-flanked area to generate Sdhafl/fl mice. IEC-specific knockout mice have been generated by intercrossing mice carrying the respective loxP-flanked alleles with Vil1-cre or Villin-creERT2 transgenic mice. Each feminine and male mice between 1 and 12 weeks of age have been utilized in all in vivo experiments, whereas metabolic tracing research have been carried out completely utilizing male mice. All mice have been maintained on the C57BL/6N background. Mice have been housed on the specific-pathogen-free animal services of the CECAD Analysis Heart of the College of Cologne beneath a 12-h darkish–12-h mild cycle in individually ventilated cages (Greenline GM500, Tecniplast) at 22 ± 2 °C and a relative humidity of 55 ± 5%. All mice had limitless entry to water and fed a regular chow eating regimen (Harlan eating regimen no. 2918 or Prolab Isopro RMH3000 5P76) advert libitum. For the experiments assessing the function of dietary fats, mice have been fed a FFD (E15104-3474, ssniff-Spezialdiäten) containing solely traces of fats (<0.5%). All animal procedures have been performed in accordance with European, nationwide and institutional pointers and protocols have been authorized by native authorities authorities (Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen) and Animal Welfare Officers of the College Medical Heart Hamburg-Eppendorf and Behörde für Gesundheit und Verbraucherschutz Hamburg. Animals requiring medical consideration have been supplied with applicable care and have been culled humanely when reaching pre-determined termination standards to reduce struggling. No different exclusion standards have been utilized. Villin-CreERT2 recombinase exercise was induced by 5 consecutive each day intraperitoneal administrations of 1 mg tamoxifen dissolved in corn oil and DMSO. Littermates not carrying the Vil1-cre or Villin-creERT2 transgenes have been used as controls in all experiments.

Tissue preparation

The colon and SI have been dissected and washed with PBS. Small items (about 0.5 cm) have been remoted proximal (after the abdomen) and distal (earlier than the caecum) of the SI, snap-frozen on dry ice for RNA expression evaluation and saved at –80 °C till additional processing. The remaining SI tissue was reduce longitudinally and washed in PBS to take away faeces. Intestinal tissue samples have been rolled up from proximal to distal to kind a Swiss roll and both fastened in 4% paraformaldehyde in a single day at 4 °C or embedded in TissueTek for frozen sectioning.

H&E staining of paraffin-fixed tissues

Paraffin-embedded 3-μm-thick intestinal tissue sections have been deparaffinized with xylene and rehydrated with reducing quantities of ethanol options (100% ethanol, 96% ethanol and 75% ethanol). Sections have been stained for two min in haematoxylin, differentiated in faucet water for 15 min and incubated for 1 min in eosin. Stained sections have been dehydrated utilizing rising quantities of ethanol options and stuck in xylene for 1 min. Slides have been mounted with Entellan.

COX and SDH and ORO staining of fresh-frozen tissues

Contemporary-frozen 7-μm-thick intestinal sections have been sequentially stained for COX and SDH exercise. Cryosections have been dried and incubated for 45 min at 37 °C with COX answer. Then they have been briefly washed with PBS and incubated for 40 min with SDH answer at 37 °C. Following dehydration by graded alcohol options, the sections have been mounted with DPX and saved at room temperature. Contemporary-frozen 10-μm-thick sections have been fastened in 4% paraformaldehyde for 15 min at room temperature. After fixation, the sections have been washed with ddH2O and stained with ORO in isopropanol/water (60:40) for 15 min. All sections have been counterstained with haematoxylin for five min and mounted with Aquatex (EMD Millipore).

Immunohistochemistry and immunofluorescence on intestinal sections

Paraffin sections have been rehydrated and heat-induced antigen retrieval was carried out in 10 mM sodium citrate, 0.05% Tween-20 at pH 6.2 or with proteinase Ok remedy. Endogenous peroxidase was blocked in peroxidase blocking buffer for 15 min at room temperature. Sections have been blocked in 1% BSA, 0.2% fish-skin gelatin, 0.2% Triton-X-100 and 0.05% Tween-20 in PBS for 1 h at room temperature. After blocking, the sections have been incubated in a single day at 4 °C with main antibodies towards adipophilin/PLIN2 (Progen, GP46, 1:500), Ki67 (Dako, M724901, clone 1O15, 1:1,000), OLFM4 (Cell Signaling, D6Y5A, clone D6X5A, 1:400), CC3 (Cell Signaling, 9661, 1:1,000), CC8 (Cell Signaling, 8592, 1:1,000), CD45 (BD Bioscience, 560510, clone 30-F11, 1:500) and F4/80 (AbD Serotec, MCA497, clone A3-1, 1:1,000). Sections have been incubated with biotinylated anti-mouse IgG (H+L) (Vector Laboratories, BA-9200-1.5, 1:1,000), anti-rabbit IgG (H+L) (Vector Laboratories, BA-1000-1.5, 1:1,000) and anti-rat IgG (H+L) (Vector Laboratories, BA-9400-1.5, 1:1,000) secondary antibodies. Every staining was visualized utilizing ABC Package Vectastain Elite (Vector, PK6100) and DAB substrate (Dako and Vector Laboratories). Immunofluorescence was carried out with main antibodies towards TGN38 (bio-techne, AF8059-SP, 1:200), E-cadherin (BD Biosciences, 610182, 1:1,000) and adipophilin/PLIN2 (Progen, GP46, 1:200). Nuclei have been stained utilizing DAPI (Vector Laboratories) and visualized with anti-sheep IgG NorthernLights NL557 (bio-techne, NL010, 1:300), anti-mouse Alexa 488 (Molecular Probes, A1101, 1:300) and anti-guinea pig Alexa 633 (Molecular Probes, A21105, 1:300) fluorescence-conjugated secondary antibodies. Periodic acid–Schiff (PAS) response was carried out in keeping with commonplace protocols. Endogenous alkaline phosphatase exercise was visualized utilizing a Quick Purple Substrate equipment in keeping with the producer’s directions (ab64254, Abcam). For picture acquisition, the intestinal sections have been analysed utilizing a light-weight microscope outfitted with a KY-F75U digital digital camera (JVC) (DM4000B, Leica Microsystems, Diskus 4.50 software program), a TCS SP8 confocal laser scanning microscope (Inverse, DMi 8 CS, Leica Microsystems LAS X, Lightning software program v.5.1.0) or a LSM Meta 710 confocal laser scanning microscope (Carl Zeiss Expertise, ZEN 2009 software program). Golgi quantification was carried out utilizing ImageJ software program (v.2.0.0.-rc-46/1.50g) as beforehand described39. The quantity and dimension of TGN38-positive fluorescent objects have been quantified utilizing the ‘analyse particles’ operate after making use of a hard and fast threshold on photos derived from maximal 2D projections of the acquired confocal stacks. Every information level corresponds to the common values from at the very least three randomly chosen intestinal areas of a single mouse. Consultant photos from 4–5 mice per genotype per time level have been analysed. Greater than 100 IEC profiles per mouse with seen nuclei have been quantified (n = 128–527).

EM evaluation

A bit of 0.5 cm proximal SI tissue was fastened in a single day in 2% glutaraldehyde (Merck) and a couple of% paraformaldehyde (Science Providers) in 0.1 M cacodylate buffer (AppliChem). Tissue samples have been handled with 1% OsO4 (Science companies) in 0.1 M cacodylate buffer for two h. After dehydration of the pattern with ascending ethanol concentrations adopted by propylene oxide, samples have been embedded in Epon (Sigma-Aldrich). Ultrathin sections (70 nm thick) have been reduce, collected onto 100 mesh copper grids (Electron Microscopy Sciences) and stained with uranyl acetate (Plano) and lead citrate (Sigma Aldrich). Photos have been captured utilizing a transmission electron microscope (Joel JEM2100 Plus) at an acceleration voltage of 80 kV, and photos have been acquired utilizing a 4K-CCD digital camera, OneView (GATAN). Mitochondrial morphological integrity quantification was carried out on randomly chosen photos of the proximal SI areas from 4 Dars2fl/fl and 4 Dars2tamIEC-KO mice. Every mitochondrial profile was categorised as regular, partly affected or severely broken primarily based on its electron density, the looks of the cristae and the extent of matrix loss (Fig. 2c). The relative distribution of the analysed mitochondria per mouse into the three morphological teams is offered. A complete of 663 mitochondrial profiles from 69 IECs versus 707 mitochondrial profiles from 80 IECs have been quantified.

Cell tradition situations and drug therapies

IEC-6 cells (ACC 111) have been bought from the Leibniz Institute DSMZ–German Assortment of Microorganisms and Cell Cultures and maintained in commonplace situations at 37 °C and 5% CO2. The cell tradition medium was composed of 45% Dulbecco’s modified Eagle medium (ThermoFisher, 41965-039), 45% RPMI 1640 (ThermoFisher, 11875093) and 0.1 U ml–1 human insulin answer (Sigma, I9278) supplemented with 10% FCS (Bio&SELL). IEC-6 cells have been routinely checked for mycoplasma contamination and examined adverse. For induction of mitochondrial dysfunction, 70–80% confluent cells have been handled for 48 h with 100 μM actinonin (A6671, Sigma-Aldrich) or 1 μM atpenin A5 (ab144194, Abcam). All compounds have been solubilized in dimethyl sulfoxide (DMSO) (A3672, PanReac AppliChem). Management cells have been handled with corresponding quantities of DMSO, which didn’t exceed 1% in tradition medium. Therapies have been renewed each 24 h. IEC-6 cells have been incubated with 5 μg ml–1 brefeldin A (B6542, Abcam) for six h. To induce LD formation, oleic acid (O1008, Sigma-Aldrich) was complexed to fatty acid-free BSA (A6003, Sigma-Aldrich) at a ratio of 6:1 and used at a focus of 600 μM after titration for twenty-four h.

Immunofluorescence of cultured cells

Immunofluorescence staining was carried out on IEC-6 cells cultured on coverslips and stuck in 4% paraformaldehyde for 15 min. Reactive aldehydes have been quenched with 50 mM NH4Cl for 10 min and the cells have been permeabilized with 0.1% Triton-X-100 in PBS for five min. After 20 min in blocking answer (0.2% fish-skin gelatin diluted in PBS), IEC-6 cells have been incubated with main antibodies towards TGN38 (bio-techne, AF8059-SP, 1:200) and MTCO1/COX1 (Molecular Probes, 459600, 1D6E1A8, 1:100) for 30 min at room temperature, adopted by incubation with anti-sheep IgG NorthernLights NL557 (bio-techne, NL010, 1:300) or anti-mouse Alexa 488 (Molecular Probes, A1101, 1:300) fluorescence-conjugated secondary antibodies for 30 min at room temperature. When LDs have been stained, 5 μM of BODIPY 493/503 (D3922, Invitrogen) diluted in PBS was utilized for 30 min. Lastly, IEC-6 cells have been mounted in Vectashield containing DAPI. For picture acquisition, a TCS SP8 confocal laser scanning microscope (Inverse, DMi 8 CS, Leica Microsystems LAS X, Lightning software program v.5.1.0) was used. Quantification of Golgi morphology was carried out utilizing ImageJ software program (v.2.0.0.-rc-46/1.50g) on 2D projections from Z-stack pictures. A complete of 4–6 randomly chosen viewing fields per situation, capturing at the very least 30 cells per picture, have been used. Golgi morphology was categorised into 5 distinct classes primarily based on TGN38-positive fluorescent objects (Prolonged Knowledge Fig. 11a,b) as follows: (1) regular (juxtanuclear Golgi ribbon composed of related stacks); (2) ring (ring-like Golgi constructions surrounding the whole nucleus); (3) condensed (bulb-shaped juxtanuclear Golgi construction); (4) fragmented (Golgi ribbon changed by extra and smaller tubules and vesicles optimistic for TGN38); and (5) dispersed (full lack of Golgi ribbon and dispersal of the TGN38 sign). Quantification was carried out by manually classifying the TGN38 sample in every cell in one of many 5 Golgi phenotypes by the identical observer, who was blinded to the experimental situations. Three unbiased experiments have been quantified.

Measurement of serum parameters

Glucose (GLU2), whole ldl cholesterol (CHOL2), triacylglycerol (TRIGL), high-density lipoprotein (HDLC4) and low-density lipoprotein (LDLC3) ranges within the blood serum from mice aged 1–12 weeks outdated have been measured utilizing commonplace assays in a Cobas C111 Biochemical Analyzer (Roche Diagnostics).

Isolation of mitochondria and evaluation of mitochondrial respiratory complexes with blue native electrophoresis

Mitochondria isolation

The SI was chopped into small items and homogenized with a rotating Teflon potter (Potter S, Sartorius; 20 strokes, 1,000 r.p.m.) in a buffer containing 100 mM sucrose, 50 mM KCl, 1 mM EDTA, 20 mM TES and 0.2% fatty acid-free BSA, pH 7.6 adopted by differential centrifugation at 850g and eight,500g for 10 min at 4 °C. Mitochondria have been washed with BSA-free buffer, and protein concentrations have been decided utilizing Bradford reagent. Mitochondria have been subjected to blue native polyacrylamide gel electrophoresis (BN-PAGE) adopted by western blot evaluation or willpower of the in gel exercise of respiratory complexes.

BN-PAGE

Mitochondrial protein concentrations have been decided utilizing Bradford reagent (Sigma). A complete of 20 μg of mitochondria was lysed for 15 min on ice in dodecylmaltoside (5 g g–1 of protein) for particular person respiratory complexes, or digitonin (6.6 g g–1 protein) for supercomplexes, and cleared from insoluble materials for 20 min at 20,000g, 4 °C. Lysates have been mixed with Coomassie G-250 (0.25% last). Mitochondrial complexes have been resolved by BN-PAGE utilizing 4–16% NativePAGE Novex Bis-Tris mini gels (Invitrogen) in a Bis-Tris/Tricine buffering system with cathode buffer initially supplemented with 0.02% G-250 adopted by the 0.002% G-250.

Complicated I in-gel exercise

Gels have been incubated in a buffer containing 0.01 mg ml–1 NADH and a couple of.5 mg ml–1 nitrotetrazolium blue in 5 mM Tris-HCl pH 7.4.

Western blot evaluation

Separated mitochondrial complexes have been transferred onto a polyvinylidene fluoride membrane utilizing a moist switch methanol-free system. Membranes have been immunodecorated with indicated antibodies adopted by ECL-based sign detection. The next antibodies have been used: anti-MTCO1 (Molecular Probes, 459600, clone 1D6E1A8, 1:5,000), anti-COX4L1 (Molecular Probes, A21348, clone 20E8C12, 1:1,000), anti-UQCRC1 (Molecular Probes, 459140, clone 16D10AD9AH5, 1:4,000), anti-NDUFS1 (Proteintech, 12444-1-AP, 1:1,000), anti-NDUFS2 (Abcam, ab96160, 1:1,000), anti-NDUFV2 (Proteintech, 15301-1-AP, 1:1,000), anti-UQCRFS1/RISP[5A5] (Abcam, ab14746, clone 5A5, 1:1,000), anti-ATP5A (Abcam, ab14748, 1:3,000), anti-SDHA (Molecular Probes, 459200, clone 2EGC12FB2AE2, 1:5,000) and anti-NDUFA9 (Molecular Probes, 459100, clone 20C11B11B11, 1:1,000).

Isolation of IECs

SI tissue was collected from mice, washed in DPBS (14190-094, Gibco) to take away faeces and reduce longitudinally. IECs have been remoted by sequential incubation of intestinal tissue in pre-heated 1 mM dithiothreitol and 1.5 mM EDTA options at 37 °C whereas shaking. Pellets of IECs have been frozen at −80 °C for additional processing.

Protein lysate preparation

IEC pellets have been lysed in RIPA lysis buffer (10 mM Tris-Cl (pH 8), 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% Triton X-100 and 0.1% SDS). Lysis buffer was supplemented with protease and phosphatase inhibitor tablets (Roche). The protein focus was measured utilizing Pierce 660 nm Protein Assay reagent (22660, Thermo Scientific) and a BSA commonplace pre-diluted set starting from 0 to 2,000 μg ml–1 (23208, Thermo Scientific). Cell lysates have been separated on SDS–PAGE and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore). A protein dimension ladder (26620, Thermo Scientific) was used for dimension comparability. Membranes have been blocked with 5% milk and 0.1% PBST and have been probed in a single day with main antibodies towards the next antibodies: DARS2 (Proteintech, 13807-1-AP, 1:1,200); whole OXPHOS rodent WB antibody cocktail (Abcam, ab110413, 1:1,000); MTCO1 (Molecular Probes, 459600, clone 1D6E1A8, 1:5,000); COX4L1 (Molecular Probes, A21348, clone 20E8C12, 1:1,000); UQCRC1 (Molecular Probes, 459140, clone 16D10AD9AH5, 1:4,000); NDUFS1 (Proteintech, 12444-1-AP, 1:1,000); NDUFS2 (Abcam, ab96160, 1:1,000); NDUFV2 (Proteintech, 15301-1-AP, 1:1,000); UQCRFS1/RISP[5A5] (Abcam, ab14746, Clone 5A5, 1:1,000); ATP5A (Abcam, ab14748, 1:3,000); SDHA (Molecular Probes, 459200, clone 2EGC12FB2AE2, 1:5,000); NDUFA9 (Molecular Probes, 459100, clone 20C11B11B11, 1:1,000); α-tubulin (Sigma Aldrich, T6074, clone TUBA4A, 1:1,000); TOMM70 (Sigma, HPA014589, 1:500); β-actin (Santa Cruz, sc-1616, clone I-19, 1:1,000); adipophilin/PLIN2 (Progen, GP46, 1:500); FABP2 (Proteintech, 21252-1-AP, 1:500); FASN (Cell Signaling, 3189S, 1:1,000); vinculin (Cell Signaling, 13901, 1:1,000); and ApoB (Beckman Coulter, 467905, 1:500). Membranes have been incubated for 1 h at room temperature with anti-rabbit IgG (GE Healthcare, NA934V, 1:5,000), anti-mouse IgG (GE Healthcare, NA931, 1:5,000), anti-goat IgG (Jackson Laboratories, 705-035-003, 1:5,000) or anti-guinea pig IgG (Progen, 90001, 1:5,000) secondary HRP-coupled antibodies and Amersham ECL Western Blotting Detection reagent (GE Healthcare) have been used. The membranes have been re-probed after incubation in Restore Western Blot stripping buffer (21059, ThermoFisher). The sign was measured with a Curix 60 Processor and a western blot imager (FUSION Solo X, Vilber).

RNA isolation from tissues

SI tissue samples have been disrupted utilizing a Precellys 24 tissue homogenizer (Bertin applied sciences). Isolation of RNA was carried out utilizing a NucleoSpin RNA isolation equipment (Macherey Nagel ref. 740955.250) in keeping with the producer’s directions.

RT–qPCR

cDNA was ready utilizing a Superscript III cDNA-synthesis equipment (18080-044, Thermo Scientific). RT–qPCR was carried out utilizing TaqMan probes (Life Applied sciences) and SYBR Inexperienced (Thermo Scientific). The mRNA expression of every gene was normalized to the expression of the housekeeping genes Tbp or Hprt1. Relative expression of gene transcripts was analysed utilizing the two–ΔΔCt technique. The RT–PCR information have been collected utilizing QuantStudio 12K Flex Software program v.1.6 (Utilized Biosystems). The next Taqman probes have been used: Olfm4 (Mm01320260_m1, Thermo Scientific), Lgr5 (Mm00438890_m1, Thermo Scientific), Ascl2 (Mm01268891_g1, Thermo Scientific), Tbp (Mm00446973_m1, Thermo Scientific), Prominin-1 (Mm00477115_m1, Thermo Scientific) and Lrig-5 (Mm00456116_m1, Thermo Scientific). Primer sequences for SYBR Inexperienced are described in Supplementary Desk 5.

C.
elegans strains, upkeep and imaging

Strains have been cultured on OP50 Escherichia coli-seeded NGM plates, in keeping with commonplace protocols40. Strains used on this examine are Bristol N2, RT1315 unc-119(ed3); pwIs503[pvha-6::mans::gfp;cbrunc-119], VS25 hjIs14 [vha-6p::GFP::C34B2.10(SP12) + unc-119(+)] and RT130 pwIs23 [vit-2::GFP]. RNAi knockdown was carried out as beforehand described41. All of the experiments have been carried out with hermaphrodite worms at days 1 and 4 of maturity that have been randomly chosen and weren’t allotted into teams. dars-2, sar-1, sec-13 and fum-1 clones have been obtained from the Ahringer RNAi library41 and confirmed by sequencing. As a management, empty L4440 vector was used. For confocal imaging, animals have been immobilized on 2% agarose pads in 5 mM levamisole buffer and imaging was carried out utilizing a spinning disc confocal microscope (Inverse, Nikon TiE, UltraView VoX, Perkin Elmer, Volocity software program). For fluorescence imaging, worms have been immobilized on 2% agarose pads in 50 mM sodium azide buffer and imaged utilizing the optical Zeiss Axio Imager Z1 microscope (ZEN 2009 software program). Photos have been analysed utilizing the open-source software program Fiji (ImageJ, v.1.53c).

RNA isolation and RT–qPCR in C.
elegans

Worms have been collected from a 9 cm plate and whole RNA was remoted utilizing Trizol (Invitrogen). DNAse remedy was carried out utilizing DNA-free, DNAse and elimination (Ambion, Life applied sciences) in keeping with the producer’s protocol. RNA was quantified by spectrophotometry and 0.8 μg of whole RNA was reverse transcribed utilizing a Excessive-Capability cDNA Reverse Transcription equipment (Utilized Biosystems). For every situation, six unbiased samples have been ready. qPCR was carried out utilizing a Step One Plus Actual-Time PCR system (Utilized Biosystems) with the next PCR situations: 3 min at 95 °C, adopted by 40 cycles of 5 s at 95 °C and 15 s at 60 °C. Amplified merchandise have been detected utilizing SYBR Inexperienced (Sensible III Extremely-Quick SYBR Inexperienced qPCR Grasp Combine, Agilent Applied sciences). Relative quantification was carried out towards Y45F10D.4.

The next primers have been used: dars-2 FW1 (5′-GTTTGCTGGGGAAATTCAGA-3′); dars-2 RV1 (5′-AGTGGAGCCGTAAATGGATG-3′); Y45F10D.4 FW (5′-GTCGCTTCAAATCAGTTCAGC-3′); and Y45F10D.4 RV (5′-GTTCTTGTCAAGTGATCCGACA-3′). Knowledge have been analysed utilizing ΔΔCt evaluation.

Lipidomics

For lipid analyses, mouse tissue samples have been homogenized in deionized water (10 μl per 1 mg moist weight) utilizing a Precellys 24 homogenizer (Peqlab) at 6,500 r.p.m. for 30 s. The protein content material of the homogenate was routinely decided utilizing bicinchoninic acid.

Liquid chromatography coupled to electrospray ionization tandem mass spectrometry

Sphingolipid (ceramides and sphingomyelins) and levels of cholesterol in mouse SI tissue have been decided by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). For sphingolipid analyses, 50 μl of tissue homogenate was used. Lipid extraction and LC–ESI-MS/MS evaluation have been carried out as beforehand described42,43. For the willpower of levels of cholesterol, 25 μl of tissue homogenate was extracted and processed as beforehand described44.

Nano-ESI-MS/MS

Ranges of cholesteryl esters (CEs), diacylglycerols (DAGs), TAGs and glycerophospholipids in mouse SI tissue have been decided by nano-ESI-MS/MS). Subsequent, 10 μl (for DAGs) or 5 μl (for TAGs and CEs) of tissue homogenate was diluted to 500 μl with Milli-Q water and combined with 1.875 ml of chloroform, methanol and 37% hydrochloric acid 5:10:0.15 (v/v/v). Subsequent, 20 μl of 4 µM d5-TG inside commonplace combination I (for TAGs), 15 μl of 256 μM CE 19:0 (for CEs) or 20 μl of 4 μM d5-DG inside commonplace mixtures I and II (for DAGs) (Avanti Polar Lipids) have been added. Lipid extraction and nano-ESI-MS/MS analyses of DAGs and TAGs have been carried out as beforehand described45. The detection of CE species was performed in positive-ion mode by scanning for precursors of m/z 369 Da at a collision vitality of 15 eV and with a declustering potential of 100 V, an entrance potential of 10 V and a cell exit potential of 14 V. Ranges of glycerophospholipids (that’s, phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylserines and phosphatidylglycerols) have been decided by performing extraction and nano-ESI-MS/MS measurement of 10 μl of tissue homogenate as beforehand described46.

Metabolomics

Metabolite extraction

Metabolite extraction answer (50% methanol, 30% acetonitrile, 20% water and 5 μM valine-d8 as inside commonplace) was added to 10–20 mg frozen SI tissue samples at an extraction ratio of 25 μl mg–1 on dry ice. Samples have been then homogenized utilizing a Precellys 24 tissue homogenizer (Bertin Applied sciences). The ensuing pattern suspension was vortexed, combined at 4 °C in a Thermomixer for 15 min at 1,500 r.p.m. after which centrifuged at 16,000g for 20 min at 4 °C. The supernatant was collected for LC–MS evaluation.

Metabolite measurement by LC–MS

LC–MS chromatographic separation of metabolites was achieved utilizing a Millipore Sequant ZIC-pHILIC analytical column (5 μm, 2.1 × 150 mm) outfitted with a 2.1 × 20 mm guard column (each 5 mm particle dimension) with a binary solvent system. Solvent A was 20 mM ammonium carbonate and 0.05% ammonium hydroxide. Solvent B was acetonitrile. The column oven and autosampler tray have been held at 40 °C and 4 °C, respectively. The chromatographic gradient was run at a stream fee of 0.200 ml min–1 as follows: 0–2 min: 80% solvent B; 2–17 min: linear gradient from 80% solvent B to twenty% solvent B; 17–17.1 min: linear gradient from 20% solvent B to 80% solvent B; 17.1–22.5 min: maintain at 80% solvent B. Samples have been randomized and analysed with LC–MS in a blinded method with an injection quantity of 5 μl. Pooled samples have been generated from an equal combination of all particular person samples and analysed interspersed at common intervals inside the pattern sequence as a high quality management. Metabolites have been measured utilizing a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (HRMS) coupled to a Dionex Final 3000 UHPLC. The mass spectrometer was operated in full-scan, polarity-switching mode, with the spray voltage set to +4.5 kV/–3.5 kV, the heated capillary held at 320 °C and the auxiliary fuel heater held at 280 °C. The sheath fuel stream was set to 25 items, the auxiliary fuel stream was set to fifteen items and the sweep fuel stream was set to 0 unit. HRMS information acquisition was carried out in a variety of m/z = 70–900, with the decision set at 70,000, the automated achieve management (AGC) goal at 1 × 106 and the utmost injection time at 120 ms. Metabolite identities have been confirmed utilizing two parameters: (1) precursor ion m/z was matched inside 5 ppm of theoretical mass predicted by the chemical system; (2) the retention time of metabolites was inside 5% of the retention time of a purified commonplace run with the identical chromatographic technique.

Knowledge evaluation

Chromatogram overview and peak space integration have been carried out utilizing the Thermo Fisher software program Tracefinder (v.5.0). The height space for every detected metabolite was subjected to the ‘Filtering 80% Rule’, half minimal lacking worth imputation and normalized towards the whole ion rely of that pattern to appropriate any variations launched from pattern dealing with by instrument evaluation. Samples have been excluded after performing testing for outliers primarily based on geometric distances of every level within the PCA rating plot as a part of the muma bundle (v.1.4)47. Afterwards, differential metabolomics evaluation was carried out. Intimately, the R bundle ‘gtools’ (v.3.8.2) (cran.r-project.org/web/packages/gtools/index.html) was used to calculate the log2(fold change) utilizing the features ‘foldchange’ and ‘foldchange2logratio’ (parameter base = 2).The corresponding P worth was calculated utilizing the R base bundle ‘stats’ (v.4.0.5) (www.r-project.org) with the operate ‘t.check’ (SIMPLIFY = F). The P worth was adjusted utilizing the stats base operate ‘p.modify’ (technique = “bonferroni”). Volcano plots have been generated utilizing the EnhancedVolcano bundle48 (v.1.8.0).

QuantSeq 3′ mRNA sequencing

RNA high quality was evaluated primarily based on the RNA integrity quantity (RIN) and OD260/280 and OD260/230 ratios. RIN values have been decided utilizing TapeStation4200 and RNA Display Tapes (Agilent Applied sciences). Gene expression was decided utilizing a QuantSeq 3′ mRNA-Seq Library Prep equipment FWD for Illumina (Lexogen). QuantSeq libraries have been sequenced on an Illumina NovaSeq 6000 sequencer utilizing Illumina RTA v.3.4.4 base-calling software program. Pattern exclusion standards have been OD260/280 < 1.8, OD260/230 < 1.5 and RIN < 7. Illumina adapters have been clipped off the uncooked reads utilizing Cutadapt with commonplace parameters and a minimal learn size of 35 after trimming (shorter reads have been discarded). QuantSeq-specific options have been intentionally not eliminated to keep away from lack of reads. Trimmed reads have been mapped to a concatenation of the mouse genome (Mus_musculus.GRCm38.dna.chromosome.*.fa.gz, downloaded from ftp.ensembl.org/pub/release-100/fasta/mus_musculus/dna/) and the ERCC92 Spike In sequences (downloaded from assets.thermofisher.com/TFS-Assets/LSG/manuals/ERCC92.zip) utilizing subread-align model v.2.0.1 with parameters -t 0 -d 50 -D 600 –multiMapping -B 5. Genomic matches have been counted utilizing featureCounts with parameters -F “GTF” -t “exon” -g “gene_id” –minOverlap 20 -M –primary -O –fraction -J -Q 30 -T 4. The genome annotation used was Mus_musculus.GRCm38.100.gtf (downloaded from ftp.ensembl.org/pub/release-100/gtf/mus_musculus/), augmented by entries for the ERCC92 Spike Ins.

All analyses have been accomplished in R-4.0.0, utilizing the performance of Bioconductor v.3.11. For differential gene expression evaluation, the bundle DESeq2 was used (bioconductor.org/packages/release/bioc/html/DESeq2.html).

Pairwise comparisons have been carried out between genotypes TG and WT (differential_expression_DESeq2_tg_VS_wt.xlsx). Genes have been excluded from a DESeq2 run if that they had a zero rely in additional than half of the samples in both of the situations in contrast. Be aware that DESeq2 units the P worth and the adjusted P worth to NA for genes with too few counts or with excessive outlier counts. Such genes have been eliminated after evaluation from the DESeq2 output.

The output tables have been augmented by gene symbols and descriptions, which have been derived from the org.Mm.eg.db annotation bundle utilizing the operate AnnotationDbi::mapIDs (bioconductor.org/packages/release/bioc/html/AnnotationDbi.html). As well as, the uncooked learn counts per gene and pattern, as returned by featureCounts, have been appended to the rows of every output desk. The statistical check producing the P values is the Wald check, and P‐adjusted values have been calculated utilizing the false discovery fee and Benjamini–Hochberg strategy. It was computed utilizing the operate nbinomWaldTest of the Bioconductor R bundle DESeq2, primarily based on a adverse binomial normal linear mannequin of the gene counts from a beforehand described technique49.

Proteomics

In-solution digestion for MS

Samples for MS evaluation have been ready by in-solution digestion. Protein (20 μg) was precipitated for at the very least 1 h in 4 volumes (v/v) of ice-cold acetone and protein pellets have been extracted by centrifugation at 13,000g for 10 min and dissolved in urea buffer (6 M urea, 2 M thiourea in 10 mM HEPES, pH 8.0). Urea-containing samples have been lowered by making use of tris(2-carboxyethyl)phosphine at a last focus of 10 mM, alkylated with chloroacetamide at a last focus of 40 mM and incubated for 1 h at room temperature. Samples have been then digested with 1 μl LysC for two h at room temperature, diluted with 50 mM ammonium bicarbonate to a urea focus of two M, incubated with 1 μl 0.5 mg ml–1 trypsin in a single day at room temperature, acidified to 1% formic acid and purified utilizing Cease and Go extraction ideas (StageTips)50.

MS-based proteome evaluation

Proteome samples have been analysed utilizing LC–MS/MS on an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher) with a FAIMS Professional system utilizing a mix of two compensation voltages of –50 V and –70 V. Chromatographic peptide separation was achieved on 50 cm reverse-phase nanoHPLC-columns (ID 75 μm, PoroShell C18 120, 2.4 μm) coupled to an EASY-nLC 1200 HPLC system and a binary buffer system A (0.1% formic acid) and B (80% acetonitrile/0.1% formic acid). Samples derived from in-solution digestion have been measured over a 120 min gradient, elevating the content material of buffer acetonitrile from 3.2 to 22% over 102 min, from 22 to 45% over 8 min and from 45 to 76% over 2 min. The column was washed with 76% acetonitrile for 8 min. Full MS spectra (300–1,750 m/z) have been recorded at a decision of 60,000, most injection time of 20 ms and computerized achieve management goal of 6 × 105. The 20 most ample ion peptides in every full MS scan have been chosen for higher-energy collisional dissociation fragmentation at nominal collisional vitality of 30. MS2 spectra have been recorded at a decision of 15,000, a most injection time of twenty-two ms and an computerized achieve management goal of 1 × 105. This MS acquisition program was alternatively run for each FAIMS compensation voltages to cowl totally different peptide fractions.

MS information processing and evaluation

The generated MS uncooked information have been analysed utilizing MaxQuant evaluation software program and the carried out Andromeda software program (v.1.6.14)51,52. Peptides and proteins have been recognized utilizing the canonical mouse UniProt database (downloaded August 2019) with frequent contaminants. All parameters in MaxQuant have been set to the default values. Trypsin was chosen because the digestion enzyme, and a most of two missed cleavages was allowed. Methionine oxidation and amino-terminal acetylation have been set as variable modifications, and carbamidomethylation of cysteines was chosen as a hard and fast modification. The label-free quantification algorithm was used to quantify the measured peptides and the ‘match between runs’ choice was enabled to quantify peptides with a lacking MS2 spectrum. Subsequent statistical evaluation was carried out utilizing Perseus (1.5.8.5) software program. Potential contaminants and reverse peptides have been excluded, and values have been log2 remodeled. Uncooked information have been assigned to 2 teams (TG and WT) and protein teams have been filtered for 4 legitimate values in at the very least one group earlier than lacking values have been changed from regular distribution (width of 0.3; down shift of 1.3). Welch’s Scholar t-test with S0 = 0.1 and a permutation-based false discovery fee of 0.01 with 500 randomizations was carried out to acquire differentially regulated proteins between the 2 teams. Recognized proteins have been annotated with the gene ontology phrases organic course of, molecular operate, and mobile compartment, and the Reactome Pathway database. Lastly, graphical visualization was achieved utilizing Prompt Clue software program53 (v.0.5.3).

GSEA and information visualization

Gene set enrichment strategies have been utilized utilizing GSEA and over-representation evaluation (ORA). Intimately, GSEA was carried out through the use of gene units revealed on the MsigDB (Reactome, KEGG, Biocarta and Hallmarks)54 and from a printed examine55 (ATF4) utilizing the packages fgsea56 (v.1.16.0) and GSEABase57 (v.1.52.1). Volcano plots have been generated utilizing the EnhancedVolcano bundle48 (v.1.8.0). The ORA was carried out utilizing the ‘enrich_GO’ operate (parameters: keyType = “ENTREZID”, OrgDb = org.Mm.eg.db, ont = “ALL”, pAdjustMethod = “BH”, qvalueCutoff = 0.1) of the clusterProfiler bundle58(v.3.16.1). The output information have been plotted utilizing the ‘emapplot’ operate of the enrichplot bundle (v. 1.8.1) (www.bioconductor.org/packages/release/bioc/html/enrichplot.html) (parameters: pie_scale = 1, showCategory = 40, format = “properly”).

Metabolic tracer research

Postprandial glucose and fats tolerance assessments

Mice have been fasted for two h earlier than receiving an oral gavage of 300 μl of a glucose–lipid emulsion containing triolein (3.6 g kg–1 physique weight), lecithin (0.36 g kg–1 physique weight) and glucose (2 g kg–1 physique weight), traced with [3H]triolein (1.4 MBq kg–1 physique weight) and [14C]DOG (1.7 MBq kg–1 physique weight). After 2 h, mice have been anaesthetized and transcardially perfused with PBS containing 10 U ml–1 heparin. Organs have been collected, weighed and dissolved in 10× (v/w) Solvable (Perkin Elmer), and radioactivity (in d.p.m.) was measured by scintillation counting utilizing a Perkin Elmer Tricarb scintillation counter. Uptake of radioactive tracers was calculated per whole organ weight.

CM manufacturing

Mice have been injected with tyloxapol (500 mg in 0.9% NaCl per kg physique weight) to dam vascular lipolysis. Mice obtained an oral gavage of a lipid emulsion with triolein (3.6 g kg–1 physique weight) and lecithin (0.36 g kg–1 physique weight) that have been traced with [14C]ldl cholesterol (1.4 MBq kg–1 physique weight) and [3H]triolein (1.7 MBq kg–1 physique weight). Blood was collected from the tail vein at 0, 30, 60 and 120 min after gavage. Plasma triglycerides have been decided by commonplace colorimetric assays (Roche) and radioactivity was measured by scintillation counting.

Plasma parameters

Plasma was generated by centrifugation of EDTA-spiked blood for 10 min at 10,000 r.p.m. at 4 °C in a bench prime centrifuge. Free glycerol was decided photometrically utilizing Free Glycerol reagent (F6428, Sigma). For lipoprotein profiling, 150 μl pooled plasma was diluted with an equal quantity of FPLC buffer (whole 300 μl), which was separated by fast-performance liquid chromatography (FPLC) on a Superose 6 10/300 GL column (GE Healthcare) with a stream fee of 0.5 ml min–1. Forty fractions (0.5 ml every) have been collected, and ldl cholesterol and triglyceride concentrations have been measured in every one.

To isolate the TRL fractions, 200 μl of plasma was combined with 200 μl density answer 1 (0.9% NaCl, 10 mM EDTA, 10 mM Tris-Cl pH 8.6 and 0.49 g ml–1 KBr; density 1.3 g l–1). The density answer 2 (0.9% NaCl, 10 mM EDTA, 10 mM Tris-Cl pH 8.6; density 1.006 g l–1) was positioned right into a Beckman TL100 centrifuge tube (Beckman, 343778) after which the plasma rigorously beneath layered. Ultracentrifugation was carried out in a Beckman Optima MAX-XP ultracentrifuge for two h at 4 °C and 40,000 r.p.m. in a Beckman TL100 rotor. After centrifugation, 200 μl of the highest containing TRL particles have been collected utilizing a syringe.

Statistical evaluation

Knowledge proven in column graphs characterize the imply ± s.e.m., as indicated within the determine captions. The D’Agostino–Pearson omnibus normality check was utilized to check regular (Gaussian) distribution. When information fulfilled the standards for normality, unpaired two-sided Scholar’s t-tests with no assumption of equal variance have been carried out; in any other case, the nonparametric Mann–Whitney U-test was chosen. A number of pairwise comparisons of teams over time by repeated measures have been evaluated by two-way ANOVA with Bonferroni’s correction for a number of comparability (the corrected P values are given for comparability between genotypes at every time level). Survival curves have been in contrast utilizing Gehan–Breslow–Wilcoxon check. The chi-squared check was used for the comparability of the mitochondria integrity distribution between two teams and the evaluation of the Golgi sample distribution after numerous inhibitor therapies in IEC-6 cells. The variety of mice analysed in every experiment is described within the respective determine captions. Statistical analyses have been carried out with GraphPad Prism 6 (v.6.01) and 9 (v.9.4.1).

Reporting abstract

Additional info on analysis design is out there within the Nature Portfolio Reporting Summary linked to this text.

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