Summary
Cancers of the gastrointestinal tract including esophageal adenocarcinomas, colorectal cancers, and cancers of the gastric cardia are common comorbidities of obesity. Excessive delivery of macronutrients to the cells lining the gut can increase one’s risk for these cancer by inducing imbalances in the rate of intestinal stem cell proliferation vs. differentiation, which can produce polyps and other aberrant growths. We demonstrate that serine palmitoyltransferase (SPT), which diverts dietary fatty and amino acids into the sphingolipid biosynthesis pathway, is a critical modulator of intestinal stem cell homeostasis. SPT and other enzymes in the biosynthetic pathway are upregulated in human colon tumors. These enzymes produce sphingolipids that serve as pro-stemness signals that stimulate peroxisome-proliferator activated receptor alpha (PPARα)-mediated induction of fatty acid binding protein-1. This increases fatty acid uptake and oxidation and enhances the stemness program. Serine palmitoyltransferase thus serves as a critical link between dietary macronutrients, epithelial regeneration, and cancer risk.
Introduction
The intestinal epithelium is the most rapidly regenerating tissue in the body. Constant mechanical damage inflicted by passing bowel content necessitates the nearly complete renewal of the epithelium every 4–5 days (Leblond and Stevens, 1948). Replacement of the intestinal villi results from a coordinated process triggered by the proliferation and differentiation of the intestinal stem cells (ISCs) that lie in a crypt at the base of the villus. The ISCs give rise to the specialized absorptive cells (i.e. enterocytes) and mucus and hormone-secreting goblet and enteroendocrine cells, respectively, that comprise the bulk of the villus epithelium. The balance between ISC proliferation and differentiation is tightly regulated and its disruption leads to gastroenteritis or hyperproliferative lesions and tumors.
ISCs respond to nutritional cues that increase proliferation and enhance the regenerative capacity of the gut epithelium. For example, the fatty acid palmitate stimulates proliferation of ISCs, thus increasing the number of cells within the crypt to enhance tumor risk (Beyaz et al., 2016). By contrast, serine deprivation restricts growth of intestinal cancers (Maddocks et al., 2017). Since palmitate and serine are required to synthesize the sphingoid backbone of sphingolipids, we developed the hypothesis tested herein that one or more sphingolipids might be important signaling intermediates linking these dietary macronutrients to stem cell fate.
Once palmitate enters cells, it is quickly conjugated to coenzyme-A, which traps the fatty acid in the cell and activates it for subsequent metabolism. The resultant palmitoyl-CoA can be coupled to (i) a glycerol backbone to produce glycerophospholipids, (ii) carnitine for transport into mitochondria, or (iii) the aforementioned amino acid serine to generate sphingolipids. This latter serine conjugation step is catalyzed by serine palmitoyltransferase (SPT), which generates the sphingoid backbone present in sphingolipids. This moiety subsequently acquires additional, variable fatty acids and a critical double bond to form ceramides, which are the precursors of sphingomyelins and gangliosides (Figure 1a). Ceramides, particularly those containing a C16-acyl chain, have emerged as important nutrient signals that alter cellular metabolism in response to excessive ectopic fatty acids (Summers et al., 2019). Though sphingolipids are relatively minor components (Figure 1B, light blue) of the mouse gut lipidome that are far less abundant than glycerolipids and sterols (Figure 1B, dark blue), the studies presented herein reveal that they are potent nutrient signals that alter metabolism of ISCs to enhance stemness and catalyze epithelial regeneration.
Results
Sphingolipids and sphingolipid-synthesizing enzymes are upregulated in human colon cancer
Adenocarcinomas that were surgically removed from colon cancer patients (Table 1) contained higher levels of C16-ceramides, C16-dihydroceramides, and sphingosine than the healthy tissue surrounding the lesion(Figure 1C). They also contained higher levels of transcripts encoding two critical SPT subunits (SPTLC1&2) and several other sphingolipid-synthesizing enzymes (i.e. CERS6, DEGS1&2, and SGMS1&2) (Figures 1D). CERS6 encodes the ceramide synthase that is required to make the C16-ceramides that are signals of nutritional excess (Hammerschmidt et al., 2019; Raichur et al., 2019; Raichur et al., 2014; Turpin et al., 2014) and are elevated in the human tumors (Figure 1C). By comparison, the other CERS isoforms (1-5), and the ceramides they produce, were unaltered in the lesions (Figure 1C and S1B). The SGMS transcripts that encode the sphingomyelin synthases that convert ceramide into sphingomyelin were slightly elevated, but sphingomyelins were not (Figure S1A). Moreover, other genes that influence ceramide synthesis or metabolism were unaltered (Figure S1B). Thus, the increase in transcripts and sphingolipids was largely restricted to the enzymes in the de novo synthesis pathway that produce the C16-ceramides. We also quantified two glycerol-containing lipids (i.e. diacylglycerol and triacylglycerol), finding that they were reduced in the tumors (Figure 1E). This finding suggests that these intestinal tumors redirect fatty acids away from the glycerolipid pathway and towards sphingolipids. As expected, the tumors contained substantially higher levels of LGR5, a marker of ISCs (Figure S1C).
Excision of Sptlc2 from mouse intestines decreases ceramides and other sphingolipid biosynthetic intermediates within the intestinal crypts
To better understand the role of sphingolipids in the gut, we produced conditional knockout mice that allow for acute depletion of the Sptlc2 subunit of SPT from intestinal epithelial cells (IECs). These Sptlc2δIEC mice were generated by breeding Sptlc2fl/fl mice that we described previously (Chaurasia et al., 2016) with mice expressing a tamoxifen inducible Cre-recombinase controlled by the villin promoter (Tg(Vil-cre/ERT2)(Goh et al., 2015). Within two days of tamoxifen administration, adult Sptlc2δIEC mice displayed a marked reduction in Sptlc2 mRNA expression in the small intestine and colon, as compared to tamoxifen-treated Sptlc2fl/fl littermates (Figure 2A). The depletion of Sptlc2 reduced levels of the sphingolipids in the de novo synthesis pathway (i.e. ceramides, dihydroceramides, sphinganine, and phytoceramides) within the intestinal crypts (Figure S2A). Surprisingly, it did not alter levels of complex sphingolipids (i.e. sphingomyelins and glucosylceramides) (Figure S2A). The effects of Sptlc2 depletion manifest predominantly in the crypts, and not the villi; tamoxifen-treatment only affected dihydroceramides, phytoceramides and sphinganine, but not other sphingolipids, within villi of the Sptlc2δIEC mice (Figure S2B).
Excision of Sptlc2 from mouse intestines disrupts gut architecture and depletes proliferating ISCs
Deletion of Sptlc2 led to rapid weight loss (Figure 2B) and death of all animals within 4 days (Figure S2F). Upon necropsy, the Sptlc2δIEC mice were found to have a short small intestine (Figure 2C and Figure S2C) and a small spleen (Figure S2D). The intestine exhibited increased permeability (Figure 2D), inflammation (Figure S2E). Treatment with broad-spectrum antibiotics prolonged animal survival (Figure S2F), suggesting that sepsis resulting from loss of the intestinal barrier was the ultimate cause of death of the animals. Careful histological assessment revealed severe disruption of the small intestine characterized by disorganized villi and nearly complete loss of crypts (Figure 2E). Following the four-day tamoxifen regimen, the Sptlc2δIEC mice showed alterations in many cell-types within the villus structure; Goblet cells (PAS), Paneth cells (LYZ1) and ISCs (OLFM4) were depleted and/or mis-localized (Figure 2F). Immunohistochemistry with anti-Ki67 antibodies also revealed a stark decrease in the rapidly proliferating cells located within the crypt (Figure 2F).
To identify which cell types were first affected by Sptlc2 depletion, we conducted a temporal assessment of the changing pathology, collecting the jejunum through each of the first three days following tamoxifen administration. Though the gross epithelial structure of small intestine in the Sptlc2δIEC mice remained relatively intact through the first three treatment days, the ISC pool started to disappear within 2-days of tamoxifen injection. This finding was evidenced by reduced expression of OLFM4 (Figure 2G), a disproportionate reduction in the number of crypts (Figure 2H), and fewer proliferating cells in the crypts as assessed by Ki67 staining (Figure S2G and S2H).
Excision of Sptlc2 from ISCs recapitulates the lethal phenotype of the SptlcδIEC mice
The findings presented thus far suggest that sphingolipids might be autonomous and essential signals that control the proliferation of ISCs in order to promote epithelial regeneration. Experiments using RNA in situ hybridization reinforced this idea, as they revealed that Sptlc2 transcript levels were much higher in the crypts than they were in the villi (Figure 2I). This observation strongly suggests that the ISCs within the crypt were the major site of sphingolipid synthesis and action. This conclusion was corroborated by the lipidomic assessments showing higher ceramide and phytoceramide levels in the crypts, as compared to the villi (Figure S3A). To selectively study the role of ISC sphingolipids, we generated a mouse line allowing for ISC-specific Sptlc2 depletion (Sptlc2δISC) by breeding the aforementioned Sptlc2fl/fl mice with Olfm4-EGFP-ires-CreERT2 mice (Schuijers et al., 2014). Injecting tamoxifen into the Sptlc2δISC mice led to depletion of Sptlc2 from the crypts, while it was retained in the other cells in the villi (Figure 2I). This led to quick depletion of the ISCs, as assessed by labeling Olfm4 using RNA in situ hybridization (Figure 2I). By comparison, Lyz1, which demarcates Paneth cells in the crypt, was largely unaffected by Sptlc2 depletion (Figure 2I). In these Sptlc2δISC mice, tamoxifen also recapitulated the gross phenotype of the Sptlc2δIEC mice, including the reduction in body weight, rapid death, short small intestine and small spleen (Figure S3B-S3F). Histologic analysis showed a comparable disruption of the small intestine structure that was most pronounced in the crypts (Figure 2J).
Restoring sphingolipids enhances stemness and promotes intestinal organoid survival
To better understand how SPT regulates stem cells, we turned to an intestinal organoid 3-D culture system (Sato et al., 2009). These mini-intestines contain all of the cell types of the mature epithelium but allow for precise control of the tissue environment. Pharmacological inhibition of SPT (i.e. treatment with the SPT inhibitor myriocin) or genetic ablation of Sptlc2 [i.e. treatment with 4-hydroxytamoxifen (4OHT)] led to rapid death of the organoids, as assessed by propidium iodide staining (Figure 3A). Thus, we recapitulated the in vivo phenotype caused by Sptlc2 depletion using this in vitro system. Remarkably, viability of the organoids could be restored by supplementing with short-chain analogs of ceramide (C2-ceramide, 25 μM) (Figure 3A). Mass spectroscopy revealed that C2-ceramide restored levels of many sphingolipids in either the myriocin or 4OHT treated organoids (e.g. ceramides, glucosylceramides, sphingomyelins and sphingosine), excepting those sphingolipids that cannot be re-produced from C2-ceramide via the salvage pathway (e.g. sphinganine and dihydroceramides) (Figure S4A and S4B). In 4OHT-treated organoids, removing C2-ceramide from the media re-initiated the death program (Figure 3A).
To clarify which sphingolipid species might be important for organoid viability, we also exposed these organoid cultures to either the glucosylceramide synthase inhibitor D-threo-et-P4 or the sphingomyelin synthase inhibitor D609, which inhibit the production of glucosylceramides and sphingomyelin, respectively. Neither of these compounds recapitulated the myriocin effects on organoid survival (Figure S4C). Collectively, these data suggest that intermediates in the de novo sphingolipid synthesis pathway (e.g. sphingosine, ceramides or phytoceramides), rather than the abundant complex sphingolipids that comprise the majority of the sphingolipidome, (e.g. sphingomyelins), were requisite for organoid viability.
To confirm that C2-ceramide modulated stemness, we quantified the number of crypt domains per organoid following exposure to C2-ceramide. As we anticipated, treating the secondary organoids with C2-ceramide decreased the number of buds sprouting from each organoid, supporting the idea that sphingolipids enhanced stemness (Figure 3B).
Ceramides enhance stemness by stimulating FABP1 expression to increase fatty acid uptake
To explore the molecular mechanism(s) that might account for the profound sphingolipid actions in the intestinal epithelium, we conducted RNA sequencing on Sptlc2-knockout organoids treated with or without C2-ceramide. Fabp1, which encodes a fatty acid binding protein (i.e. FABP1) involved in lipid uptake, was amongst the handful of transcripts most dramatically upregulated by C2-ceramide (Figure 3C). This C2-ceramide effect on FABP1 was particularly interesting owing to a bevy of studies showing that saturated fatty acids increase the expression of FABP1 in ISCs (Mihaylova et al., 2018) and that increasing fatty acid oxidation enhances stemness (Bensard et al., 2019; Knobloch et al., 2017; Mihaylova et al., 2018). Sptlc2 depletion from Sptlc2δIEC mice decreased Fabp1 transcripts in the crypt, but not the villi (Figure 3D). Using organoids, we also confirmed that Sptlc2 depletion decreased, and C2-ceramide restored, expression of Fabp1 mRNA and FABP1 protein (Figure 3E and 3F).
To evaluate the functional consequences of these interventions on lipid handling, we quantified fatty acid uptake in organoids isolated from the Sptlc2δIEC mice treated with vehicle, 4OHT, or C2-ceramide. C2-ceramide stimulated fatty acid uptake in organoids. By contrast, depletion of Sptlc2 using 4OHT reduced rates of fatty acid uptake (Figure 3G).
The RNA sequencing also identified the transcript encoding carnitine palmitoyltransferase-1 (Cpt1a), the enzyme that facilitates fatty acid entry into mitochondria, as a C2-ceramide-responsive gene. We confirmed that C2-ceramide slightly increased expression of Cpt1a transcripts (by qPCR, Figure 4A) and CPT1a protein (by Western blot, Figure 4B). The CPT1 inhibitor etomoxir negated the protective actions of C2-ceramide on survival of Sptlc2 knockout organoids (Figure 3H). Thus, induction of fatty acid uptake and utilization were essential C2-ceramide actions that preserved ISC viability.
Ceramides increase FABP1 expression through PPARα activation
Prior studies have shown that the transcription factors peroxisome-proliferator activated receptor isoforms alpha (PPARα) and delta (PPARδ) regulate FABP1 expression in the small intestine (Darimont et al., 1998; Hughes et al., 2015; Mochizuki et al., 2001; Poirier et al., 2001). In addition, recent studies have implicated PPARδ as a critical intermediate linking saturated fats to the control of fatty acid oxidation and the enhancement of ISC stemness (Beyaz et al., 2016; Mihaylova et al., 2018). We thus tested whether Fabp1 or Cpt1 expression was controlled by either of these transcription factors. The PPARα antagonist GW6471 blocked the C2-ceramide induction of Fabp1 (Figure 4A and 4B), but not Cpt1a. The PPARδ antagonist GSK 3787 had no effect on either gene (Figure 4A). Thus, PPARα, but not PPARδ, was an essential intermediate linking C2-ceramide to the induction of FABP1.
Lastly, we evaluated whether PPARα was sufficient to maintain organoid viability, even in the absence of Sptlc2. The PPARα agonist GW7647 restored viability of organoids lacking Sptlc2 (Figure 4C and 4D). GW7647-treated organoids had fewer crypts in each organoid, thus recapitulating the C2-ceramide effect on organoid stemness (Figure 4E and 4F). Moreover, PPARα activation recapitulated the ceramide effect on Fabp1 expression (Figure 4G). These studies convincingly display the existence of a sphingolipid-PPARα-FABP1 axis that links macronutrients to the maintenance of the ISC pool.
Discussion
These studies provide important mechanistic insight about the nutritional signals that control the metabolism and proliferation of ISCs, the cell-of-origin of gastrointestinal cancers. Prior studies have shown that the transition from normal ISCs into a hyperplastic lesion is dependent upon fuel choice (Bensard et al., 2020). Excessive fatty acid supply increases the proliferation and stemness of ISC and progenitors within the crypt to promote tumorigenicity (Bensard et al., 2019; Knobloch et al., 2017; Mihaylova et al., 2018). In mouse models of colorectal cancer, genetic depletion or pharmacological inhibition of the mitochondrial pyruvate carrier, which increases dependence on fatty acids oxidation, expands the stem cell compartment and doubles the frequency of adenoma formation (Bensard et al., 2020; Schell et al., 2017). By contrast, germline deletion of Fabp1 slows lipid uptake and reduces adenoma formation (Dharmarajan et al., 2013). The findings described herein reveal that sphingolipids produced from ectopic palmitate and serine influence this fuel choice by enhancing fatty acid import through FABP1.
Our data further identify PPARα as an essential intermediate that links endogenous sphingolipids to the induction of Fabp1. This is consistent with prior studies, which show that high fat diet increases levels of Pparα and its downstream target Fabp1 in rat jejunum (Suruga et al., 1999), as well as those showing that a PPARα antagonist induces organoid death (Stine et al., 2019). Moreover, it aligns with studies showing that sphingolipids increase PPARα-mediated transcription (Correnti et al., 2018; Murakami et al., 2011; Tsuji et al., 2009; Van Veldhoven et al., 2000). Ceramides, phytoceramides, and sphingoid bases have been identified as upstream activators of PPARα, with some synthetic sphingolipids showing direct binding capabilities (Murakami et al., 2011; Tsuji et al., 2009; Van Veldhoven et al., 2000). Our findings reveal that one or more of these sphingolipids serves as a signal to increase fatty acid uptake and oxidation, and thus provides important new information about the nutritional sensing machinery that induces this transcriptional program.
The studies are also important for discerning the precise role of SPT in the gut. Prior studies have shown that genetic ablation of Sptlc2 (Li et al., 2018; Ohta et al., 2009) or pharmacological inhibition of SPT (Genin et al., 2016) has deleterious consequences on gut health, with the former causing animal death and the latter gastric enteropathy. Indeed, inflammatory bowel disease has been associated with decreased expression of SPT (Li et al., 2018). The studies described herein indicate that these pathogenic states result from decreased conversion of free fatty acid and amino acids into ceramides and a concomitant depletion of the ISC pool. Thus, strategies targeting sphingolipids could prove effective as a means of modulating the health of the intestinal barrier.
Collectively, these data support the conclusion that overproduction of sphingolipids enhances stemness of cells within the intestinal crypts. Using the organoid system, we identified an intriguing mechanism linking sphingolipids to the modulation of stemness through their effects on PPARα-mediated induction of FABP1 and a resultant increase in fatty acid uptake. These observations help reveal how saturated fatty acids control intestinal stemness and provide an explanation for the utility of serine deprivation as a means to decrease tumor incidence. Finally, because stem cells play a major role not only in tissue regeneration but also in carcinogenesis, deciphering their response to dietary factors is of the utmost importance for the treatment of degenerative conditions. Ultimately, these studies could suggest dietary or pharmacological strategies for maximizing regenerative capacity while also minimizing the risk of cancer development.
Author contributions
S.A.S, B.C., and Y.L. conceived of the project designed the experiments and wrote the manuscript. Y.L, B.C, V.A.K., J.A.M., performed experiments and analyzed data. C.B, J.R, H.C, Y.H provided some of the mouse strains used in this study. J.L.W and W.L.H. assisted in quantification of images. D.L provided human tissues used in this study and J.C, P.W and P.J.M aided in lipid analysis.
Funding
Mass spectrometry equipment for the Metabolomics core was obtained through NCRR shared instrumentation grants 1S10OD016232-01, 1S10OD018210-01A1 and 1S10OD021505-01 and microscopy equipment for histology was obtained using a NCRR Shared Equipment Grant # 1S10RR024761-01. Preclinical Research Resource was supported by the National Cancer Institute of the National Institutes of Health under Award Number P30CA042014. The authors received research support from the National Institutes of Health (DK115824, DK116888, and DK116450 to SAS; DK124326 to B.C; and DK108833 and DK112826 to WLH), the Juvenile Diabetes Research Foundation (JDRF 3-SRA-2019-768-A-B to SAS and JDRF 3-SRA-2019-768-A-B to WLH), the American Diabetes Association (to SAS), the American Heart Association (to SAS), the Margolis Foundation (to SAS), and the United States Department of Agriculture (2019-67018-29250 to B.C.). J.L.W. received support by the National Institutes of Health through the Ruth L. Kirschstein National Research Service Award 5T32DK091317 from the National Institute of Diabetes and Digestive and Kidney Diseases.
Declaration of Interests
SAS is a founder and shareholder with Centaurus Therapeutics.
Materials and Methods
Human colon samples
Human colon cancer tissues were obtained from the Preclinical Research Resource at the Huntsman Cancer Institute. Tumors were collected during tumor resection (not biopsies) and cryopreserved as viable tissue. The adjacent normal tissue was collected as a paired control.
Animal Colonies
All animal experiments were conducted with protocols approved by the Institutional Animal Care and Use Committee (IAVUC) of the University of Utah. Generation of Sptlc2δIEC Mice. Sptlc2fl/fl mice were crossed with Villin-Cre-ERT2 mice(Goh et al., 2015) to generate gut-specific Sptlc2 knockout animals. To deplete the gene, mice were injected intraperitoneally with tamoxifen (1mg/mouse) every day for three days at 8-10 weeks of age. Sptlc2fl/fl mice littermates that were also injected with tamoxifen were used as controls. Generation of Sptlc2δISC Mice. Sptlc2fl/fl mice were crossed with Olfm4-EGFP-ires-CreERT2 mice (26) to generate ISC-specific Sptlc2 knockout animals. To deplete the gene, mice were injected intraperitoneally with tamoxifen (3mg/mouse) every day for five days at 8-10 weeks of age. Sptlc2fl/fl mice littermates that were also injected with tamoxifen were used as controls.
Sphingolipidomics
University of Utah Metabolomics Core
Tissues or organoids suspended in 120 ◻1 PBS went through three freeze-thaw cycles to homogenize the samples. Thereafter, 100 ◻1 of homogenate was used for lipid extraction and 20 ◻1 was saved for protein assays. Samples were spiked with 225 μL MeOH containing internal standards (deuterated ceramide mixture (C16, 60 pmol/sample; C18, 35 pmol/sample; C24, 150 pmol/sample; C24:1, 312 pmol/sample); dihydroceramide C18:1, 5pmol/sample; deuterated sphingomyelin, 50 pmol/sample; glucosylceramide C17, 50 pmol/sample; and deuterated S1P, 5pmol/sample) followed by 750 μL MTBE (methyl tert-butyl ether). The samples were then incubated for 60 min on ice with brief vortexing every 15 min. Following centrifugation for phase separation, the upper phase was transferred to another Eppendorf tube which was dried using a Speedvac. The dried, extracted lipid was reconstituted in methanol:toluene (9:1). The lipid extracts were then analyzed by liquid chromatography-mass spectrometry (LC/MS). Briefly, lipid extracts were separated on an Acquity UPLC CSH C18 1.7 μm 2.1 × 50 mm column maintained at 60 °C connected to an Agilent HiP 1290 Sampler, Agilent 1290 Infinity pump, and Agilent 6490 triple quadrupole (QqQ) mass spectrometer. Sphingolipids were detected using dynamic multiple reaction monitoring (dMRM) in positive ion mode. Source gas temperature was set to 210°C, with a gas (N2) flow of 11 L/min and a nebulizer pressure of 30 psi. Sheath gas temperature was 400°C, sheath gas (N2) flow of 12 L/min, capillary voltage is 4000 V, nozzle voltage 500 V, high pressure RF 190 V and low-pressure RF is 120 V. Injection volume was 2 μL and the samples were analyzed in a randomized order with the pooled QC sample injection eight times throughout the sample queue. Mobile phase A consisted of ACN:H2O (60:40 v/v) and mobile phase B consists of IPA:ACN:H2O (90:9:1 v/v) both in 10 mM ammonium formate and 0.1% formic acid. The chromatography gradient started at 15% mobile phase B, increased to 30% B over 1 min, increased to 60% B from 1-2 min, increased to 80% B from 2-10 min, and increased to 99% B from 10-10.2 min where it was held until 14 min. Post-time was 5 min and the flowrate was 0.35 mL/min throughout. Collision energies and cell accelerator voltages were optimized using sphingolipid standards with dMRM transitions as [M+H]+→[m/z = 284.3] for dihydroceramides, [M+H]+→[m/z = 287.3] for isotope labeled dihydroceramides, [M-H2O+H]+→[m/z = 264.2] for ceramides, [M-H2O+H]+→[m/z = 267.2] for isotope labeled ceramides and [M+H]+→[M-H2O+H]+ for all targets. Sphingolipids without available standards were identified based on HR-LC/MS, quasi-molecular ion and characteristic product ions. Their retention times were either taken from HR-LC/MS data or inferred from the available sphingolipid standards. Results from LC/MS experiments are collected using Agilent Mass Hunter Workstation and analyzed using the software package Agilent Mass Hunter Quant B.07.00. Sphingolipids were quantitated based on peak area ratios to the standards added to the extracts.
Baker Heart and Diabetes Institute Metabolomics Laboratory
The quantification of specific lipids at the Baker Heart and Diabetes Institute Metabolomics laboratory was done as follows: 200 μL of chloroform/methanol (2:1, v/v) were added together with 10 μL ISTD in chloroform/methanol (1:1, v/v) to 25-60μg of protein lysate in 10μl cold PBS. The mixture was mixed for 10 min on a rotary mixer, sonicated in a water bath (18 °C–24 °C) for 30 min, left to stand on the bench for 20 min and then centrifuged (16,000 × g, 10 min, 20 °C). The supernatant was transferred to a 96-well plate and dried under a stream of nitrogen gas at 40 °C. Samples were reconstituted with 50 μL H2O-saturated 1-butanol and sonicated for 10 min. Finally, 50 μL of 10 mM ammonium formate in MeOH was added. The extract was centrifuged (1700 × g, 5 min, 20 °C) and the supernatant was transferred into a 0.2 mL glass insert with Teflon insert cap for analysis by LC ESI-MS/MS as described (27).
Isolation, culture, and analysis of intestinal organoids
Small intestinal crypts were isolated and grown into organoids as described previously(Sato et al., 2009). Isolated small intestines were chopped into small pieces and washed in cold PBS. They were then incubated in 2 mM EDTA with PBS for 30 min on ice. EDTA medium was removed and the tissue fragments were washed by cold PBS. They were then pipetted vigorously to dissociate and enrich the crypts into the supernatant. Isolated crypts were pull down by centrifuged at 200g, and then re-suspended in Matrigel (Car#354277, BD) for organoids culture. Myriocin (10uM) was used to inhibit SPT2 activity in the organoids for 24-48 hours. Deletion of Sptlc2 in organoid cultures from Sptlc2μIEC mice was induced by addition of 200ng/ml 4-OH-tamoxifen (4-OHT) to the culture medium for 48 hours. Cell death of organoid cultures was determined by propidium iodine (PI)-positive staining. Images were taken by EVOS digital inverted microscope. Quantification of PI staining was done by Image J software (NIH).
Antibiotic treatment of Sptlc2δIEC mice
A broad-spectrum antibiotic mixture was added to the drinking water one month before the ablation of the Sptlc2 gene(Takahashi et al., 2014). The cocktail included 200mg ciprofloxacin (Sigma-Aldrich), 1g ampicillin (Sigma-Aldrich), 100mg metronidazole (Sigma-Aldrich), 500mg vancomycin (Labconsult), 1g neomycin, 2.50g streptomycin (ThermoFisher), 100,000U penicillin (ThermoFisher), 1g bacitracin (Sigma-Aldrich) and 1g ceftazidime (Sigma-Aldrich) were dissolved in 1 liter of water. Feces was collected and cultured in liquid cultures of Thioglycollate medium (Sigma) and McConkey agar plates. No bacteria growth was observed from both cultures.
Quantitative RT-PCR
Total RNA was extracted from tissues or cells using the RNeasy Mini Isolation Kits with DNA digestion (QIAGEN) according to the manufacturer’s instructions. Isolated total RNA was reverse-transcribed into cDNA using commercially available kits (Quantbio). All subsequent qRT-PCR reactions were performed on a QuantStudio 12K Flex Real-Time PCR system (Thermo Fisher Scientific) using the Qiagen QuantiFast SYBR Green PCR kit. qPCR primers were purchased from Sigma-Aldrich, KiCqStart Primers. For normalization threshold cycles (Ct-values) of all replicate analyses were normalized to Ppib or B2M for mouse tissues and human tissue within each sample to obtain sample-specific ΔCt values (= Ct gene of interest - Ct Ppib, B2M). To compare the effect of various treatments with untreated controls, 2−ΔΔCt values were calculated to obtain fold expression levels, where ΔΔCt = (ΔCt treatment - ΔCt control).
FITC-Dextran Permeability Test
Intestinal permeability was assessed by oral gavage of FITC–dextran (Sigma-Aldrich). Mice were administered 500◻ FITC-Dextran (20mg/ml in PBS, 10mg/mouse) by gavage. Serum sample were collected 4-hours after oral gavage; Fluorescence were measured by a fluorescence plate reader at 488nm. Serum FITC-Dextran concentration was calculated according to the standard curve made by a serial dilution from the stock in PBS.
RNAseq Analysis
Total RNA was extracted and RNAseq analysis was conducted at University of Utah genomics core facility who evaluated the RNA quality by NanoDrop spectrophotometer and Agilent Bioanalyzer RNA 6000 Chip and further performed the sequencing by Illumina HiSeq2000 with 50 cycle paired end sequencing. Data were analyzed by bioinformatics analysis service at the microarray core facility at the University of Utah. The criteria for differential expression included only genes with FDR of <0.05 or log-transformed false discovery rate (FDR) of >13 and with a fold change greater than or equal to ±1.5 relative to controls. The Fragments Per Kilobase Million (FPKM) values of the differentially expressed genes were log transformed and normalized to the average of the controls. These values were used to produce hierarchical clustering and generate heat maps of the genes using Cluster 3.0 and Java Tree View open source programs, respectfully.
Western Blot Analysis
Proteins were extracted from organoids by homogenizing in RIPA buffer (0.5% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5) containing protease inhibitors (Complete Mini, Roche). The homogenate was cleared by centrifugation at 4°C for 30 min at 15,000 g and the supernatant containing the protein fraction recovered. Protein concentration in the supernatant was determined using the BCA Protein Assay Kit (Pierce). 10 μg of proteins was resolved using precast Bolt (4-12% Bis-Tris gels, Invitrogen) and transferred to Nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% BSA in Tris-buffered saline containing 0.2% Tween-20 (TBS-T) and incubated with primary antibodies at 4°C overnight.
Histology and Immunochemistry
For histology, tissues were fixed in 10% buffered formalin for 48 hours, embedded in paraffin, sectioned at 5μm and mounted on the slides. The slides were either stained with hematoxylin and eosin or PAS for goblet cells. Immunohistochemistry: The slides were sent to Arup laboratories for immunohistochemical analysis of Olfm4, Lyz1, Ki67, F4/80 and cleaved caspase-3 staining.
Fatty Acid Uptake Assay
Organoids were grown and treated with vehicle, 4OHT (200ng/ml) or C2-ceramide for 48 hours. Organoids were then dissociated into single cell suspension and seeded onto a 384-well plate. BODIPY-dodecanoic acid fluorescent fatty acid analog was added (QBT™ Fatty Acid Uptake Assay Kit, Molecular Devices) and the plate was immediately transferred to a fluorescence microplate reader for kinetic reading (every 20 seconds for 30-60 minutes) using a bottom-read mode. Measurement was done with an excitation filter of 480 nm and an emission filter of 520.
RNA fluorescence in situ hybridization
Formalin-fixed paraffin-embedded (FFPE) mouse jejunum sections were de-paraffinized, rehydrated, and then hybridized with mRNA probes against mouse Sptlc2, Olfm4 and Lyz1 in accordance with the manufacturer’s instructions (Advanced Cell Diagnostics, Newark, CA). In brief, FFPE sections were pretreated with hydrogen peroxide, incubated in Target Retrieval solution in a steamer for 30 minutes, and permeabilized by incubating in Protease Plus solution for 40 minutes. After hybridization, a fluorescent kit was used to amplify the mRNA signal. TSA Plus Fluorescein, TSA Plus Cyanine 3, and TSA Plus Cyanine 5 fluorescent signals were detected using EVOS digital inverted microscope.
Statistical analysis
Data were plotted as the mean ± SEM. Student t-test, one-way or two-way ANOVA were carried out using Excel or prism and statistical significance was considered meaningful at p<0.05.
Acknowledgements
We wish to acknowledge the support from Metabolomics, Histology, and Metabolic Phenotyping Cores at the Health Sciences Center of the University of Utah. All data are available in the main text or the supplementary materials.