DGAT2 partially compensates for lipid-induced ER stress in human DGAT1-deficient intestinal stem cells
2019; Elsevier BV; Volume: 60; Issue: 10 Linguagem: Inglês
10.1194/jlr.m094201
ISSN1539-7262
AutoresJorik M. van Rijn, Marliek van Hoesel, Cecilia de Heus, AnkeH.M. van Vugt, Judith Klumperman, EdwardE.S. Nieuwenhuis, RoderickH.J. Houwen, Sabine Middendorp,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoDietary lipids are taken up as FAs by the intestinal epithelium and converted by diacylglycerol acyltransferase (DGAT) enzymes into triglycerides, which are packaged in chylomicrons or stored in cytoplasmic lipid droplets (LDs). DGAT1-deficient patients suffer from vomiting, diarrhea, and protein losing enteropathy, illustrating the importance of this process to intestinal homeostasis. Previously, we have shown that DGAT1 deficiency causes decreased LD formation and resistance to unsaturated FA lipotoxicity in patient-derived intestinal organoids. However, LD formation was not completely abolished in patient-derived organoids, suggesting the presence of an alternative mechanism for LD formation. Here, we show an unexpected role for DGAT2 in lipid metabolism, as DGAT2 partially compensates for LD formation and lipotoxicity in DGAT1-deficient intestinal stem cells. Furthermore, we show that (un)saturated FA-induced lipotoxicity is mediated by ER stress. More importantly, we demonstrate that overexpression of DGAT2 fully compensates for the loss of DGAT1 in organoids, indicating that induced DGAT2 expression in patient cells may serve as a therapeutic target in the future. Dietary lipids are taken up as FAs by the intestinal epithelium and converted by diacylglycerol acyltransferase (DGAT) enzymes into triglycerides, which are packaged in chylomicrons or stored in cytoplasmic lipid droplets (LDs). DGAT1-deficient patients suffer from vomiting, diarrhea, and protein losing enteropathy, illustrating the importance of this process to intestinal homeostasis. Previously, we have shown that DGAT1 deficiency causes decreased LD formation and resistance to unsaturated FA lipotoxicity in patient-derived intestinal organoids. However, LD formation was not completely abolished in patient-derived organoids, suggesting the presence of an alternative mechanism for LD formation. Here, we show an unexpected role for DGAT2 in lipid metabolism, as DGAT2 partially compensates for LD formation and lipotoxicity in DGAT1-deficient intestinal stem cells. Furthermore, we show that (un)saturated FA-induced lipotoxicity is mediated by ER stress. More importantly, we demonstrate that overexpression of DGAT2 fully compensates for the loss of DGAT1 in organoids, indicating that induced DGAT2 expression in patient cells may serve as a therapeutic target in the future. Erratum: DGAT2 partially compensates for lipid-induced ER stress in human DGAT1-deficient intestinal stem cellsJournal of Lipid ResearchVol. 62PreviewVOL 60 (2019) PAGES 1787–1800 Full-Text PDF Open Access Interest in the systemic response to dietary lipids has gained attention, as the global spread of a Western lifestyle has led to a vast increase in the occurrence of obesity (1Kopelman P.G. Obesity as a medical problem.Nature. 2000; 404: 635-643Crossref PubMed Scopus (3652) Google Scholar). This trend has a significant impact on global healthcare, as obesity is strongly correlated with an increased risk of a wide variety of health problems, including diabetes, heart disease, and colorectal cancer (2Renehan A.G. Tyson M. Egger M. Heller R.F. Zwahlen M. Body-mass index and incidence of cancer: a systematic review and meta-analysis of prospective observational studies.Lancet. 2008; 371: 569-578Abstract Full Text Full Text PDF PubMed Scopus (3696) Google Scholar, 3Li R. Grimm S.A. Mav D. Gu H. Djukovic D. Shah R. Merrick B.A. Raftery D. Wade P.A. Transcriptome and DNA methylome analysis in a mouse model of diet-induced obesity predicts increased risk of colorectal cancer.Cell Rep. 2018; 22: 624-637Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Although much research has focused on the systemic effects of a high-fat diet (HFD), much remains unknown about how the intestinal epithelium copes with lipid-induced stress as the primary port of entry for dietary lipids. Lipids in the human diet consist mainly of triglycerides (TGs), which comprise a mixture of saturated FAs (SFAs) and unsaturated FAs (UFAs). In the intestine, these hydrophobic TGs are emulsified and subsequently hydrolyzed by lipases into monoglycerides and FFAs. Diacylglycerol acyltransferase (DGAT)1, located on the ER, finally catalyzes the synthesis of TG from fatty acyl-CoA and diacylglycerol (DG). The resulting hydrophobic TGs are either packaged in chylomicrons (CMs) for export into the lymph or stored in cytosolic lipid droplets (LDs) (4Yen C.L. Nelson D.W. Yen M.I. Intestinal triacylglycerol synthesis in fat absorption and systemic energy metabolism.J. Lipid Res. 2015; 56: 489-501Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Interest in this process has increased in the last decade because the intestine has been found to store dietary lipids locally for delayed release up to 16 h post-meal (5Chavez-Jauregui R.N. Mattes R.D. Parks E.J. Dynamics of fat absorption and effect of sham feeding on postprandial lipema.Gastroenterology. 2010; 139: 1538-1548Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The intestine is therefore potentially an important regulating organ for systemic lipid metabolism, instead of solely a port of entry. Furthermore, it has been shown in other cell types, including murine fibroblasts and adipocytes, that DGAT1 is involved in the formation of LDs, which protect these tissues from lipid-induced stress upon high FA load (6Welte, M. A., and A. P. Gould, . Lipid droplet functions beyond energy storage. Biochim. Biophys. Acta Mol. Cell Biol Lipids. 1862: 1260–1272.Google Scholar, 7Nguyen, T. B., S. M. Louie, J. R. Daniele, Q. Tran, A. Dillin, R. Zoncu, D. K. Nomura, and J. A. Olzmann, . DGAT1-dependent lipid droplet biogenesis protects mitochondrial function during starvation-induced autophagy. Dev. Cell. 42: 9–21.e5.Google Scholar, 8Chitraju C. Mejhert N. Haas J.T. Diaz-Ramirez L.G. Grueter C.A. Imbriglio J.E. Pinto S. Koliwad S.K. Walther T.C. Farese Jr., R.V. Triglyceride synthesis by DGAT1 protects adipocytes from lipid-induced ER stress during lipolysis.Cell Metab. 2017; 26: 407-418.e3Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 9Olzmann J.A. Carvalho P. Dynamics and functions of lipid droplets.Nat. Rev. Mol. Cell Biol. 2019; 20: 137-155Crossref PubMed Scopus (793) Google Scholar). Recently, we have shown that patients deficient for DGAT1 have severe clinical features, such as protein losing enteropathy (PLE), vomiting and/or diarrhea, fat intolerance, and failure to thrive due to impaired fat metabolism, but fare well on a lipid-restricted diet (10van Rijn J.M. Ardy R.C. Kuloğlu Z. Härter B. van Haaften-Visser D.Y. van der Doef H.P.J. van Hoesel M. Kansu A. van Vugt A.H.M. Thian M. Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency.Gastroenterology. 2018; 155: 130-143.e15Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). As PLE in combination with diarrhea can be the result of mucosal injury in the intestine (11Braamskamp M.J.A.M Dolman K.M. Tabbers M.M. Clinical practice: protein-losing enteropathy in children.Eur. J. Pediatr. 2010; 169: 1179-1185Crossref PubMed Scopus (108) Google Scholar), the concurrent fat intolerance indicated that DGAT1 protects the epithelium from lipid-induced damage. By using patient-derived dermal fibroblasts and intestinal organoids, we have shown that DGAT1 mediates LD formation, and DGAT1 deficiency increases sensitivity to lipid-induced toxicity and apoptosis of intestinal epithelial cells. However, LD formation was not completely abolished in patient-derived DGAT1-deficient cells upon stimulation with the most common UFA, oleic acid (OA) (10van Rijn J.M. Ardy R.C. Kuloğlu Z. Härter B. van Haaften-Visser D.Y. van der Doef H.P.J. van Hoesel M. Kansu A. van Vugt A.H.M. Thian M. Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency.Gastroenterology. 2018; 155: 130-143.e15Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). So far, it was not resolved how LD formation was established in the absence of DGAT1, or by what mechanism LD formation protects the epithelium against lipotoxicity. The availability of patient-derived intestinal organoids provides a unique opportunity to study LD-mediated protection against lipotoxicity specifically in human intestinal epithelial cells. The DGAT1-independent LD forming capacity we found in patient-derived intestinal organoids hints toward the presence of an endogenous rescue mechanism, and therewith to a potential target for treatment of DGAT1-deficient patients. As DGAT2, an isozyme of DGAT1, was previously described to contribute to LD formation in other cell types and organisms (12Kuerschner L. Moessinger C. Thiele C. Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets.Traffic. 2008; 9: 338-352Crossref PubMed Scopus (304) Google Scholar, 13Stone S.J. Levin M.C. Zhou P. Han J. Walther T.C. Farese R.V. The endoplasmic reticulum enzyme DGAT2 is found in mitochondria-associated membranes and has a mitochondrial targeting signal that promotes its association with mitochondria.J. Biol. Chem. 2009; 284: 5352-5361Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 14Wilfling F. Wang H. Haas J.T. Krahmer N. Gould T.J. Uchida A. Cheng J.X. Graham M. Christiano R. Fröhlich F. et al.Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets.Dev. Cell. 2013; 24: 384-399Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar), we hypothesized that the DGAT1-independent LD formation we have observed previously was mediated by DGAT2 activity. Here, we determined whether DGAT2 has a role in LD formation and by which mechanism lipid-induced cytotoxicity is mediated in DGAT1-deficient intestinal organoids. As such, we determined the effects of both DGAT1- and DGAT2-mediated lipid metabolism on the homeostasis of the intestinal epithelium. We show that epithelial stem cells express both functional DGAT1 and DGAT2, despite the fact that DGAT2 was previously shown to be expressed only in very low levels in human intestine (15Haas J.T. Winter H.S. Lim E. Kirby A. Blumenstiel B. DeFelice M. Gabriel S. Jalas C. Branski D. Grueter C.A. et al.DGAT1 mutation is linked to a congenital diarrheal disorder.J. Clin. Invest. 2012; 122: 4680-4684Crossref PubMed Scopus (108) Google Scholar, 16Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members.J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). Furthermore, we show that functional DGAT2 expression is lost upon differentiation toward an enterocyte phenotype, and DGAT2 can partially compensate for LD formation and resistance to lipotoxicity when DGAT1 function is inhibited in intestinal stem cells. In addition, we show that DGAT1-dependent OA-induced LD formation is required for tolerance to the SFA, palmitic acid (PA), in human intestinal stem cells, which was also partially dependent on DGAT2 in the absence of DGAT1. Furthermore, we link the protective effect of LDs to diminished lipid-induced ER stress. Finally, we show that overexpression of DGAT2 fully rescued OA-mediated lipotoxicity in patient-derived DGAT1-deficient intestinal cells. Together, our data indicate that DGAT enzymes are of crucial importance to protect the intestine from toxic concentrations of FAs. Furthermore, our results indicate that DGAT2 could have a functional role in the human intestinal stem cell niche and may serve as a therapeutic target to treat diseases related to intestinal lipid uptake. This study was approved by the responsible local ethics committee (Institutional Review Board of the University Medical Center Utrecht). All participants provided written informed consent for the collection of samples and subsequent analysis. The study was conducted in accordance with the ethical principles set forth in the Declaration of Helsinki. All organoid cultures were derived from duodenal intestinal biopsies. The control and patient groups were each made up of three individuals, referred to as control or patient 1, 2, or 3. Control 1 was a 4-year-old male at the time the biopsy was taken, control 2 a 4-year-old female, and control 3 a 9-year-old female. Extensive clinical details for each patient are provided in (10van Rijn J.M. Ardy R.C. Kuloğlu Z. Härter B. van Haaften-Visser D.Y. van der Doef H.P.J. van Hoesel M. Kansu A. van Vugt A.H.M. Thian M. Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency.Gastroenterology. 2018; 155: 130-143.e15Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), where patients 1, 2, and 3 in this study are referred to as patients 7, 8, and 9, respectively, in our previous study. Patients 1 and 2 are brothers from a consanguineous Dutch family, respectively 12 and 15 years old at the time the biopsy was taken. Patient 3 was a 9-year-old female from a Dutch family of unknown consanguinity and sister of a monozygotic twin pair. The human SI epithelium control used for Western blot was isolated from an ileum resection from a newborn male infant. Crypts were isolated from biopsies as described previously (17Wiegerinck C.L. Janecke A.R. Schneeberger K. Vogel G.F. van Haaften-Visser D.Y. Escher J.C. Adam R. Thöni C.E. Pfaller K. Jordan A.J. et al.Loss of syntaxin 3 causes variant microvillus inclusion disease.Gastroenterology. 2014; 147: 65-68.e10Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 18Sato T. Stange D.E. Ferrante M. Vries R.G.J. Van Es J.H. Van den Brink S. Van Houdt W.J. Pronk A. Van Gorp J. Siersema P.D. et al.Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (2109) Google Scholar) and grown into organoids as described previously (10van Rijn J.M. Ardy R.C. Kuloğlu Z. Härter B. van Haaften-Visser D.Y. van der Doef H.P.J. van Hoesel M. Kansu A. van Vugt A.H.M. Thian M. Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency.Gastroenterology. 2018; 155: 130-143.e15Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In short, crypts were isolated from intestinal biopsies by dissociation with 10 mM EDTA in PBS and collected by pipetting vigorously. Isolated crypts were resuspended in growth factor-free medium, consisting of Advanced DMEM/F12 (Gibco), 100 U/ml penicillin-streptomycin (Gibco), 10 mM HEPES (Gibco), and GlutaMAX (Gibco). Matrigel (Corning) was added to a final concentration of 70% and plated on prewarmed cell culture 24- or 96-well plates. After Matrigel polymerization, organoid culture medium (hSI-EM) was added consisting of growth factor-free medium, 50% WNT3A-conditioned medium, 20% Rspondin-1-conditioned medium, 10% Noggin-conditioned medium, 50 ng/ml murine EGF (Peprotech, London, UK), 10 mM nicotinamide (Sigma-Aldrich), 1.25 mM N-acetyl (Sigma-Aldrich), B27 (Gibco), 500 nM TGF-β inhibitor A83-01 (Tocris, Bristol, UK), 10 μM P38 inhibitor SB202190 (Sigma-Aldrich). Organoids were cultured at 37°C and 5% CO2, and medium was refreshed every 2–3 days. The organoids were passaged as single cells using TrypLE Express (Thermo Fisher Scientific), counted, and seeded as 250 cells/μl in the Matrigel mix. After passaging, 10 μM Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor Y-27632 (Abcam, Cambridge, UK) was added for the first 2–3 days of the culture. To induce differentiation, organoids were cultured in differentiation medium (hSI-DM) for 5 days, which consists of hSI-EM lacking WNT3A-conditioned medium, nicotinamide, and SB202190. The LD assays were performed as described previously (10van Rijn J.M. Ardy R.C. Kuloğlu Z. Härter B. van Haaften-Visser D.Y. van der Doef H.P.J. van Hoesel M. Kansu A. van Vugt A.H.M. Thian M. Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency.Gastroenterology. 2018; 155: 130-143.e15Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In short, OA-BSA conjugate was prepared by heating 0.2 g OA (Sigma-Aldrich) to 70°C in 1.5 ml PBS. The OA suspension was then thoroughly vortexed and slowly added to a solution of FA-free BSA (Sigma-Aldrich) in PBS kept at 37°C in a molar ratio of OA:BSA (8:1) to a final stock concentration of 20 mM OA and 2.5 mM BSA. PA (Sigma-Aldrich) was first saponified to 100 mM in 0.1 M NaOH and kept at 70°C for 1 h before conjugation to BSA in a final stock concentration of 10 mM in an 8:1 ratio as described for OA. Organoids were grown in expansion medium (EM) on tissue culture-treated 96-well plates. After 5 days of EM or 5 days of differentiation on differentiation medium (DM), the organoids were incubated with FA-BSA for 17 h in the presence or absence of 0.1 μM DGAT1 inhibitor (D1i) (AZD 3988; Tocris) for EM, 1 μM D1i for DM, and/or 14 nM DGAT2 inhibitor (D2i) (PF-06424439; Sigma-Aldrich). Organoids were then fixed in 4% formaldehyde for 30 min at room temperature. Cells were washed in PBS and stained with 0.025 mg/ml LD540 and DAPI (Sigma-Aldrich) in PBS for 15 min at room temperature in the dark. Cells were washed and stored in PBS. Imaging of the organoids was performed using a Leica SP8X laser-scanning confocal microscope outfitted with a white light laser. The acquired stacks were processed and analyzed with Fiji/ImageJ (19Rueden C.T. Schindelin J. Hiner M.C. DeZonia B.E. Walter A.E. Arena E.T. Eliceiri K.W. ImageJ2: ImageJ for the next generation of scientific image data.BMC Bioinformatics. 2017; 18: 529Crossref PubMed Scopus (3241) Google Scholar, 20Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. Preibisch S. Rueden C. Saalfeld S. Schmid B. et al.Fiji: an open-source platform for biological-image analysis.Nat. Methods. 2012; 9: 676-682Crossref PubMed Scopus (30534) Google Scholar); shown are maximum projections of approximately 10 micron stacks. For the flow cytometric analysis, organoids were grown and treated with OA-BSA in the same manner as for the confocal analysis. After incubation with OA-BSA, the cells were harvested by pipetting and dissociated using TrypLE Express (Thermo Fisher) until single cells were acquired. The cells were then fixed and stained in the same manner as for the confocal analysis and assayed using a BD FACS Canto II. The results were analyzed using FlowJo® software and statistical analysis was performed using GraphPad Prism 7 software. Organoids were lysed in Laemmli buffer [0.12 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.05 g/l bromophenol blue, 35 mM β-mercaptoethanol] and incubated at 100°C for 5 min. Human ileum resection material was gently scraped with a scalpel and used as the ex vivo control sample. The protein concentration was measured using a BCA assay. Equal amounts of protein were separated by SDS-PAGE on a 12% acrylamide gel and transferred to a polyvinylidene difluoride membrane using a Trans-Blot® Turbo according to manufacturer's protocol. The membrane was then blocked with 5% milk protein in TBST [0.3% Tween, 10 mM Tris-HCl (pH 8), and 150 mM NaCl in distilled water] and probed with primary antibodies. The membranes were washed with TBST and incubated with appropriate secondary antibodies. Immunocomplexes were detected using the LI-COR Odyssey. The primary antibodies used were rabbit anti-DGAT1 (1:1,000, ab181180; Abcam), rabbit anti-DGAT2 (1:1,000, bs-12998R; Bioss Antibodies), mouse anti-ACTB (sc-47778; Santa Cruz Biotechnology). The secondary antibodies used were donkey anti-rabbit IgG IRDye 680 (1:20,000, 926-32222.; LI-COR) and donkey anti-mouse IgG IRDye 800RD (1:20,000, 926-32222; LI-COR). Band intensities were quantified using Fiji/ImageJ. Within lanes, band intensities for DGAT1 and DGAT2 were normalized to ACTB. RNA was isolated using TRIzol® LS reagent (Thermo Fisher) from organoids grown in either EM for 10 days or EM for 5 days and subsequently DM for 5 days according to the manufacturer's protocol. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad) and amplified using SYBR green supermix (Bio-Rad) in a Light Cycler96® (Bio-Rad) according to the manufacturer's protocol. The comparative Ct method was used to quantify the data. The relative quantity was defined as 2−ddCt. Primers are listed in supplemental Table S3, HP1BP3 was used as housekeeper gene. For quantification of (un)spliced X-box binding protein 1 (XBP1), we performed a PCR for XBP1 using GoTaq® DNA polymerase (Promega) and Green GoTaq® Flexi buffer (Promega) with 20 ng template according to the manufacturer's protocol. The PCR was performed on a Bio-Rad T100 Thermo Cycler at 62°C for 30 cycles, the product run on a 1% agarose gel, and visualized using SYBR Safe DNA Gel stain (Thermo Fisher). Intestine organoids of patient cells and healthy control cells were fixed at room temperature by adding Karnovsky fixative [2.5% glutaraldehyde and 2% formaldehyde (Electron Microscopy Sciences) in 0.1 M phosphate buffer (pH 7.4)] 1:1 to the cell culture medium for 10 min. This was replaced by fresh fixative solution and incubated for 2 h at room temperature. Cells were then postfixed for 2 h at 4°C with 1% OsO4/1.5% K3Fe(III)(CN)6 in 0.065 M phosphate buffer and finally 1 h with 0.5% uranyl acetate in demi water. After fixation, cells were dehydrated and embedded in Epon epoxy resin (Polysciences). Ultrathin sections of 60 nm were contrasted with uranyl acetate and lead citrate using the AC20 (Leica) and examined with a T12 electron microscope (Thermo Fisher). Images were collected from three control and three patient organoid lines per experimental condition. All the measurements were done using ImageJ. LD number and area quantification was done using the freehand tool. This was done for 20 cells per sample and a total of 60 cells per condition. This dataset was cleaned using R, where "null" measurements for the LD area were removed. Subsequently, the average number of LDs per cell was calculated for 60 cells per condition, and statistical analysis was performed with an unpaired t-test using GraphPad Prism. The size of individual LDs was presented as a density plot using the ggplot2 package for R. The organoid lipotoxicity assays were performed as described previously (10van Rijn J.M. Ardy R.C. Kuloğlu Z. Härter B. van Haaften-Visser D.Y. van der Doef H.P.J. van Hoesel M. Kansu A. van Vugt A.H.M. Thian M. Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency.Gastroenterology. 2018; 155: 130-143.e15Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Briefly, for the propidium iodide (PI) staining, organoids were grown and incubated with varying concentrations of FA as described for the LD confocal assay. The organoids were then stained with Hoechst 33342 (1 μg/ml) (Sigma-Aldrich) and PI (0.1 mg/ml) (Thermo Fisher) in DMEM without phenol red (Gibco) at room temperature for 15 min. Organoids were imaged using a Zeiss LSM800 confocal microscope. Resulting images were analyzed using Fiji/ImageJ (19Rueden C.T. Schindelin J. Hiner M.C. DeZonia B.E. Walter A.E. Arena E.T. Eliceiri K.W. ImageJ2: ImageJ for the next generation of scientific image data.BMC Bioinformatics. 2017; 18: 529Crossref PubMed Scopus (3241) Google Scholar, 20Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. Preibisch S. Rueden C. Saalfeld S. Schmid B. et al.Fiji: an open-source platform for biological-image analysis.Nat. Methods. 2012; 9: 676-682Crossref PubMed Scopus (30534) Google Scholar) for the total area of fluorescence of PI. To calculate the percentage of maximum assay response, the total area of PI fluorescence was normalized to the total live and dead cell signal per well according to the formula Rx = Px/(Px + Hx) where P is the total area of PI fluorescence and H the total area of Hoechst fluorescence for FA concentration x, where x ranges [0, …, n]. Then, the percentage of maximum assay response was calculated as (Rx – R0)/[max(Rx) – R0] × 100%. The resulting percentage of total area response was averaged across technical duplicates for each line of intestinal organoids, and three biological replicates were included in the groups control and patient. Percentages of total assay response for the three biological replicates were plotted against the log10([FA]) using GraphPad Prism and fitted with a nonlinear regression with variable slope. Where applicable, the concentration resulting in half-maximal cell lethality (LC50) is defined as 10log10([FA]), giving the concentration of FA where 50% of total assay response is reached. In TABLE 1, TABLE 2, values are reported as LC50 = mean [95% CI].TABLE 1Quantification of organoid resistance to OA toxicity as represented in Fig. 4ConditionEMDMControlPatientControlPatientOA6.3 [6.0–6.5]A2.8 [2.6–3.0]a>10ND6.0 [5.5–6.5]aOA + D1i3.3 [3.1–3.4]B2.8 [2.6–3.0]a5.3 [4.6–6.0]A5.9 [5.3–6.6]aOA + D2i6.5 [6.3–6.7]A2.5 [2.3–2.8]a,b>10ND5.5 [5.2–5.9]aOA + D1i + D2i2.6 [2.3–2.8]C2.4 [2.2–2.6]b4.4 [3.7–5.2]A5.6 [5.2–6.1]aValues are shown as the LC50 values with 95% confidence interval in brackets, transformed from the log10 regression results into a linear scale, resulting in OA concentrations (millimolar) where 50% of organoids are PI positive. Cells with different letters (i.e., A, a, etc.) within experiments in EM or DM are significantly different (P < 0.05). ND, LC50 was not determined, and statistical interpretation was not possible. Open table in a new tab TABLE 2Quantification of organoid resistance to PA toxicity as represented in Fig. 5ConditionEMControlPatientPA1.10 [1.08–1.12]a,b1.17 [1.12–1.23]aPA + D2i1.12 [1.09–1.16]a,b1.06 [1.01–1.12]bPA + OA2.41 [2.37–2.46]c2.00 [1.91–2.10]dPA + OA + D2i2.28 [2.19–2.37]c1.82 [1.73–1.91]eValues are shown as the LC50 values with 95% confidence interval in brackets, transformed from the log10 regression results into a linear scale, resulting in PA concentrations (mM) where 50% of organoids are PI positive. Cells with different letters (i.e., a, b, etc.) are significantly different (P < 0.05). Open table in a new tab Values are shown as the LC50 values with 95% confidence interval in brackets, transformed from the log10 regression results into a linear scale, resulting in OA concentrations (millimolar) where 50% of organoids are PI positive. Cells with different letters (i.e., A, a, etc.) within experiments in EM or DM are significantly different (P < 0.05). ND, LC50 was not determined, and statistical interpretation was not possible. Values are shown as the LC50 values with 95% confidence interval in brackets, transformed from the log10 regression results into a linear scale, resulting in PA concentrations (mM) where 50% of organoids are PI positive. Cells with different letters (i.e., a, b, etc.) are significantly different (P < 0.05). DGAT2 overexpressing (D2OE) intestinal organoids were generated by transduction with a lentiviral overexpression construct. A lentiviral vector was constructed by cloning the DGAT2 complementary sequence from HEK293T cDNA with primers containing AgeI and XbaI restriction sites and subsequent T4 ligation into the pLenti-CMV-GFP-Hygro plasmid (21Campeau E. Ruhl V.E. Rodier F. Smith C.L. Rahmberg B.L. Fuss J.O. Campisi J. Yaswen P. Cooper P.K. Kaufman P.D. A versatile viral system for expression and depletion of proteins in mammalian cells.PLoS One. 2009; 4: e6529Crossref PubMed Scopus (591) Google Scholar) (supplemental Fig. S5). The DGAT2 stop-codon was kept in this construct, and thus it does not produce a GFP fusion protein, to avoid cross-excitation between GFP and LD540 in the lipotoxicity assay. This vector was transfected using polyethylenimine into HEK293T packaging cells at 80% confluence together with psPAX2 and pMD2G. Cell culture supernatant from the packaging cells was collected 48 and 72 h posttransfection, filtered through a 0.2 μm filter, and concentrated by ultracentrifugation at 50,000 g for 90 min. The viral pellet was resuspended in infection medium consisting of EM (10 μM Y-27632) and 8 μg/ml polybrene and stored at −80°C. Prior to transduction, healthy control and DGAT1-deficient organoids were grown in EM for 2–3 days. The organoids (2/24 wells) were then dissociated into single cells using TrypLE and resuspended in 250 μl infection medium in a 48-well suspension cell culture plate. Virus transduction was performed as reported previously (22Koo, B. K., D. E. Stange, T. Sato, W. Karthaus, H. F. Farin, M. Huch, J. H. van Es, and H. Clevers, . Controlled gene expression in primary Lgr5 organoid cultures. Nat. Methods. 9: 81–83.Google Scholar). In brief, 10 μl of high-titer virus was added to the cell suspension and mixed by gentle pipetting. The plate was centrifuged at 450 g at 32°C for 1 h. The plate was then incubated at 37°C for an additional 6 h. The cells were spun down and resuspended in 2:1 Matrigel with EM, seeded into two wells of a 24-well tissue culture plate, and grown in EM + 10 μM Y-27632. Three days after transduction, the medium was changed to EM with 100 μg/ml hygromycin B. When growth in hygromycin-resistant organoids was apparent, the organoids were split and cultured in EM. Data are presented as mean ± SD. Statistical significance was evaluated by paired two-way ANOVA within families with Tukey's post hoc (Fig. 1B, C) or Bonferroni's post hoc (Figs. 2B; 4D, H), unpaired two-tailed Student's t-test (Fig. 3E), unpaired two-way ANOVA across families with Tukey's post hoc (Fig. 5D), and paired two-way ANOVA for simple comparisons to the baseline within fami
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