Abstract
Gut microbes are linked to host metabolism, but specific mechanisms remain to be uncovered. Ceramides, a type of sphingolipid (SL), have been implicated in the development of a range of metabolic disorders from insulin resistance (IR) to hepatic steatosis. SLs are obtained from the diet and generated by de novo synthesis in mammalian tissues. Another potential, but unexplored, source of mammalian SLs is production by Bacteroidetes, the dominant phylum of the gut microbiome. Genomes of Bacteroides spp. and their relatives encode serine palmitoyltransfease (SPT), allowing them to produce SLs. Here, we explore the contribution of SL-production by gut Bacteroides to host SL homeostasis. In human cell culture, bacterial SLs are processed by host SL-metabolic pathways. In mouse models, Bacteroides-derived lipids transfer to host epithelial tissue and the hepatic portal vein. Administration of B. thetaiotaomicron to mice, but not an SPT-deficient strain, reduces de novo SL production and increases liver ceramides. These results indicate that gut-derived bacterial SLs affect host lipid metabolism.
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Introduction
Sphingolipids (SLs) are important structural and bioactive signaling molecules in mammals, with significant roles in the development of metabolic disorders1,2,3. Ceramides, a subtype of SL, are the best-studied SL in insulin resistance (IR)4,5,6. In animal models, reduction of ceramide (d18:1/16:0) in the liver alleviates IR2,7,51. Analysis of the expression of SL processing genes was done with a set of 107 manually curated genes from gene ontology categories involved in SL, ceramide, sphingosine, or sphingomyelin metabolism and are detailed in Supplementary Data 1. For heatmaps, expression values were averaged across replicates and genes with a greater than twofold change in normalized expression values are visualized in Fig. 2h.
Animal experiments
All experiments involving animals were performed according to Protocol #2010-0065 approved by the Cornell University Institutional Animal Care and Use Committee. Mice were maintained on a 12-h light/dark cycle and housed at constant temperature and humidity. All gavages of bacterial cultures were prepared from overnight cultures. Cells were centrifuged, washed and resuspended in sterile PBS and administered in a volume of 0.2 mL at a concentration of 108 CFU/mL unless otherwise noted.
Transfer of labeled bacterial lipids to mouse epithelial cells
Five to six-week-old female germfree Swiss Webster (GF SW) (Taconic Biosciences) were purchased and shipped to Cornell University, where they were allowed to acclimate for 48 h in their shipper. Upon removal from the shipper, mice were either immediately sacrificed or gavaged with overnight cultures of BTWT grown in MMG supplemented with 25 µM PAA. Mice were then housed 3–4 mice per cage and fed an autoclaved breeder diet (5021 LabDiet) ad libitum. Mice were gavaged daily with the same dose of PAA-labeled BTWT for 4 additional days until sacrifice. On the day of sacrifice, mice were fasted for 6 h then gavaged with PAA-labeled BTWT (as above) 1 h before sacrifice. Intestinal tissue was harvested for confocal imaging as described below.
Alkyne containing metabolites in bacterial cells (plated on UV-sterilized #1.5 glass coverslips in 6-well plates) were labeled with Alexa Fluor 647 using the Click iT cell reaction buffer kit (Thermo Fisher Scientific). Bacterial cells were mounted onto glass slides and imaged on an LSM 710 confocal microscope (Zeiss).
At the time of sacrifice, small intestinal tissue was cut into three equal sections. Then, 4 cm of tissue from the duodenum, jejunum, ileum, in addition to the whole length of the colon were embedded in O.C.T media and snap-frozen using isopentane and liquid N2. Blocks were cryosectioned into 8 µm sections, set and fixed on a glass slide using 4% paraformaldehyde. Slides were stained using the Click iT cell reaction buffer kit (ThermoFisher Scientific) and imaged on an LSM 710 confocal microscope (Zeiss) at the Cornell University Biotechnology Resource Center.
Hepatic portal vein blood SLs
To assess intestinal SL uptake, 5-week-GF SW mice (Taconic Biosciences) were allowed to acclimate for 2 days in their germfree ship** container. After this acclimation period, mice were either immediately sacrificed or gavaged with BTWT cultures grown overnight in MMG. Mice were housed in sterile filter top plastic cages under specific pathogen free (SPF) conditions and sacrificed 6 h post-gavage on day 1 and 7 of the experiment. Blood was collected from the hepatic portal vein by euthanizing the mice using CO2 followed by cervical dislocation. Heparin (100 µL–30 IU/mL, Sigma) was added to the cavity before nicking the hepatic portal vein and blood was collected using a Pasteur pipet. Hepatic portal vein blood was frozen in liquid N2 and stored at −80 °C.
Monoassociation of GF mice with BTWT or SLMUT
GF SW mice were bred in-house and caged in rigid sterile isolators. Mice (females, 3–4- week old) were transferred to flexible bubble isolators and inoculated with either BTWT or SLMUT by oral gavage. Mice were housed 3–4 per cage, fed an autoclaved breeder diet (5021, LabDiet) ad libitum and sterility was checked biweekly. Mice were sacrificed 6 weeks after inoculation. After decapitation, livers, PBS flushed ileum tissue, and cecal contents were collected, flash frozen in liquid N2 and stored at −80 °C until processed for SL analysis. Colonization efficiency was monitored by determining the colony forming units per gram (CFU/g) of cecal content of sacrificed mice. 50 mg of cecal content per sample (seven samples per condition) was weighted and added to 1 mL of BHIS. Serial dilutions (1:10) of the slurry were made to the dilution 1:108. Serial dilutions were plated on BHIS agar plates and incubated at 37 °C in an anaerobic chamber overnight before counting colonies.
Mice used in hepatic SL profiling
Germfree: GF SW mice (female) were bred in-house and caged in rigid sterile isolators. GF mice were sacrificed at 5 weeks of age. Conventionalized mice: 5-week-old female GF SW mice were inoculated with 0.2 mL of fecal slurry made from three mouse pellets homogenized in 3 mL of sterile PBS. Pellets were obtained from SPF Swiss Webster mice housed in the conventional corridor of the Cornell Mouse Facility. Conventionalized mice were housed in sterile filter top cages and sacrificed a week after colonization. Conventionally raised: 5-week old conventionally raised female mice were obtained from litters two generations after breeding pairs from the germfree colony had been conventionalized.
For all mice: after euthanasia by decapitation, livers were collected, flash frozen in liquid N2 and stored at −80 °C until processed for SL analysis.
Induction of hepatic de novo SL synthesis
Five-week-old female SW mice (Taconic Biosciences) were fed either a breeder (5021 LabDiet: calories from protein—23%, carbohydrates—53%, fat—24%) or a fatty acid free diet (TD.03314 Teklad: calories from protein—24%, carbohydrates—76%, fat—0%) ad libitum for 21 days while housed in SPF conditions with 2–5 mice per cage. Mice were gavaged with BTWT or SLMUT 24 h before sacrifice, and then again prior to fasting (6 h before sacrifice). After decapitation, livers were collected, flash frozen in liquid N2 and stored at −80 °C until processed for SL analysis.
Insulin-resistant mice administered BTWT or SLMUT
Seven-week-old male C57BL/6J mice (Jackson Laboratory) on a chow diet (Diet 5001, LabDiet) were gavaged with BTWT or SLMUT for 7 days and then placed on a high-fat diet (HFD; D12492, Research diet, Inc.). Mice were then gavaged for 7 additional days with BTWT and SLMUT and kept on the HFD for an additional 21 days. When mice were then switched back to a chow diet and gavaged daily with BTWT or SLMUT for 9 days before sacrifice. All gavages were 0.2 mL of cultures of BTWT or Δ0870-SLMUT (108 CFU/mL) grown in BHIS and washed in PBS. Mice were euthanized by cervical dislocation and livers were frozen in liquid N2 until processed for SL analysis.
Lipid extractions
Liver, ileum, and colon tissue were all homogenized in PBS using tubes with sterile 1 mm zirconium beads (OPS diagnostics) in a bead beater homogenizer (BioSpec products) for 2 min. Intestinal tissue was placed on ice and then subjected to another round of bead beating to ensure homogeneity of the sample. Protein concentrations of homogenates were measured using a Lowry protein assay (BioRad) and equal concentrations of samples were loaded on to 96-well plates for lipid extractions. Liver samples were extracted in 1:1 dichoromethane:methanol according to the details below with 400–800 µg of protein per sample while colon and ileum samples were extracted with 100–200 µg of protein per sample. Hepatic portal vein whole blood (150 µL) was loaded on to 96-well plates for lipid extraction.
Cecal tissues were weighed before lipids were extracted according to the Folch method52 in 4 mL of 2:1 chloroform:methanol. After 2 h of constant vortexing on a plate vortexer, 800 µL of 0.9% sodium chloride in water solution was added to the extraction. Samples were briefly vortexed again before spinning samples at 2000 × g for 15 min to separate the aqueous and organic phases. The lower organic phase was then transferred to a new tube where 800 µL of 0.9 % sodium chloride was added in order to ensure an organic phase free of cecal debris. These extractions were spun again at 2000 × g for 15 min and the organic phase was transferred to a glass tube and dried under nitrogen gas. The remaining lipids were weighed and resuspended at an equal concentration for subsequent mass spectrometry analysis.
Cell pellets were resuspended in 100 µL of PBS and protein concentrations were assessed using the Lowry method. Equal amounts of cell suspension as measured by protein concentration (100–150 µg) were loaded onto 96-well plates for SL extractions. Bacterial cell pellets were washed and resuspended in PBS and protein concentrations were assessed using the Lowry method. Equal amounts of cell suspension as measured by protein concentration were loaded onto 96-well plates for SL extractions.
All samples loaded onto 96-well plates had 50 µL of 1 µM C12 ceramide (d18:1/12:0) (Avanti Polar Lipids) added as an internal standard and 50 µL of 10% diethylamide diluted in methanol. For the lipid extraction, 450 µL of 1:1 dichloromethane:methanol was added to each sample and vortexed overnight on a plate shaker. After the overnight incubation, an additional 900 µL of 1:1 dichloromethane:methanol was added to the samples and incubated on a plate rotator for an additional hour before spinning samples at 2000 × g for 15 min to separate cell debris from the lipid extracts. The supernatant was transferred to a new 96-well plate for analysis by mass spectrometry as detailed below.
SL measures: acid base hydrolysis
Blood samples and microbial cultures were broken down to their SL long chain base backbones using harsh acid and base treatment to obtain estimates of total SL levels using the methods outlined in ref. 53. In brief, equal volume of hepatic portal vein blood (150 µL per experiment) or protein normalized microbial cultures were added to 500 µL of methanol supplemented with 4 µM of 1-deoxysphinganine-D3 (C18H36D3NO) (Avanti Polar Lipids) and vortexed on a plate vortexer for 1 h. Samples were then spun down at 21,130 × g in a microcentrifuge for 5 min to remove cell debris. Supernatants were transferred to polypropylene tubes and 75 µL of concentrated hydrochloric acid was added before incubating the samples overnight (16–20 h) at 65 °C. After overnight incubation, concentrated potassium chloride (10 M) was added to samples and a lipid extraction using chloroform was used to extract hydrolyzed SLs. The organic phase of the lipid extraction was dried under nitrogen gas and resuspended in 200 µL of 1:1 dichloromethane:methanol before adding C12 ceramide (d18:1/12:0) (Avanti Polar Lipids) internal standard to samples on a 96-well plate and analyzing SL levels by mass spectrometry as detailed below.
Lipidomic profiling
SL abundance was measured by liquid chromatography-mass spectrometry (LC-MS) using methods modified from Bui et al.54. Specifically, 4 µl of lipid extract from each sample was injected into an Agilent 1200 HPLC (Agilent Poroshell 120 column) linked to an Agilent 6430 triple quadrupole mass spectrometer. Mobile phase A consisted of methanol/water/chloroform/formic acid (55:40:5:0.4 v/v); Mobile phase B consists of methanol/acetonitrile/chloroform/formic acid (48:48:4:0.4 v/v). After pre-equilibration for 6 sec, the gradient gradually increases to 60% mobile phase B and 100% mobile phase B that is held for 1.9 min. Flow rate was 0.6 mL/min. The duration of a run was 9.65 min. Ions were fragmented using electrospray ionization in positive mode and selective reaction monitoring (SRM) allowed for the detection of SL specific transitions. The method was validated for SLs listed in Supplementary Table 3. Peak calls and abundance calculations were done using MassHunter Workstation Software Version B.06.00 SP01/Build 6.0.388.1 (Agilent). Final concentrations of samples were calculated from a standard curve for each SL (Supplementary Table 3). For Sa (d17:0) base metabolites, no standard curve was available and the response of the mass spectrometer normalized to the standard (Ceramide (d18:1/12:0)) was used for abundance calculations.
Statistical analyses
All data are represented as the mean ± SEM unless otherwise noted. Statistical tests are denoted in figure legends and were implemented using Prism 8.3.0 (GraphPad) or using the Tukey C, nlme, and multcomp packages implemented in R.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Data availability
Data for Fig. 2h and Supplementary Fig. 3 have been deposited in the Sequence Read Archive under the accession number PRJNA613677. Data underlying Figs. 1–5 and Supplementary Figs. 1, 2, 4, 8 are provided as Source Data files. All other data are available as supplementary materials or from the corresponding author upon request.
References
Raichur, S. et al. CerS2 haploinsufficiency inhibits beta-oxidation and confers susceptibility to diet-induced steatohepatitis and insulin resistance. Cell Metab. 20, 687–695 (2014).
Turpin, S. M. et al. Obesity-induced CerS6-dependent C16:0 ceramide production promotes weight gain and glucose intolerance. Cell Metab. 20, 678–686 (2014).
Hannun, Y. A. & Obeid, L. M. Principles of bioactive lipid signalling: lessons from sphingolipids. Nat. Rev. Mol. Cell Biol. 9, 139–150 (2008).
Meikle, P. J. & Summers, S. A. Sphingolipids and phospholipids in insulin resistance and related metabolic disorders. Nat. Rev. Endocrinol. 13, 79–91 (2017).
Hannun, Y. A. & Obeid, L. M. Sphingolipids and their metabolism in physiology and disease. Nat. Rev. Mol. Cell Biol. 19, 175 (2017).
Summers, S. A., Chaurasia, B. & Holland, W. L. Metabolic messengers: ceramides. Nat. Metab. 1, 1051–1058 (2019).
Chaurasia, B. et al. Targeting a ceramide double bond improves insulin resistance and hepatic steatosis. Science 365, 386–392 (2019).
**a, J. Y. et al. Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis. Cell Metab. 22, 266–278 (2015).
**e, C. et al. An intestinal farnesoid X receptor-ceramide signaling axis modulates hepatic gluconeogenesis in mice. Diabetes 66, 613–626 (2017).
Apostolopoulou, M. et al. Specific hepatic sphingolipids relate to insulin resistance, oxidative stress, and inflammation in nonalcoholic steatohepatitis. Diabetes Care 41, 1235–1243 (2018).
Lemaitre, R. N. et al. Circulating sphingolipids, insulin, HOMA-IR, and HOMA-B: The Strong Heart Family Study. Diabetes 67, 1663–1672 (2018).
Luukkonen, P. K. et al. Hepatic ceramides dissociate steatosis and insulin resistance in patients with non-alcoholic fatty liver disease. J. Hepatol. 64, 1167–1175 (2016).
Duan, R. D. Physiological functions and clinical implications of sphingolipids in the gut. J. Dig. Dis. 12, 60–70 (2011).
Chung, R. W. et al. Dietary sphingomyelin lowers hepatic lipid levels and inhibits intestinal cholesterol absorption in high-fat-fed mice. PLoS ONE 8, e55949 (2013).
Norris, G. H., Porter, C. M., Jiang, C., Millar, C. L. & Blesso, C. N. Dietary sphingomyelin attenuates hepatic steatosis and adipose tissue inflammation in high-fat-diet-induced obese mice. J. Nutr. Biochem. 40, 36–43 (2017).
Yunoki, K. et al. Dietary sphingolipids ameliorate disorders of lipid metabolism in Zucker fatty rats. J. Agric. Food Chem. 58, 7030–7035 (2010).
Heaver, S. L., Johnson, E. L. & Ley, R. E. Sphingolipids in host-microbial interactions. Curr. Opin. Microbiol. 43, 92–99 (2018).
Stoffel, W., Dittmar, K. & Wilmes, R. Sphingolipid metabolism in Bacteroideaceae. Hoppe Seylers Z. Physiol. Chem. 356, 715–725 (1975).
Karlsson, K. A. On the chemistry and occurrence of sphingolipid long-chain bases. Chem. Phys. Lipids 5, 6–43 (1970).
Merrill, A. H. Jr., Wang, E. & Mullins, R. E. Kinetics of long-chain (sphingoid) base biosynthesis in intact LM cells: effects of varying the extracellular concentrations of serine and fatty acid precursors of this pathway. Biochemistry 27, 340–345 (1988).
An, D. et al. Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells. Cell 156, 123–133 (2014).
Brown, E. M. et al. Bacteroides-derived sphingolipids are critical for maintaining intestinal homeostasis and symbiosis. Cell Host Microbe 25, 668–680 e667 (2019).
Goodman, A. L. et al. Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe 6, 279–289 (2009).
Wieland Brown, L. C. et al. Production of alpha-galactosylceramide by a prominent member of the human gut microbiota. PLoS Biol. 11, e1001610 (2013).
Cohen, L. J. et al. Commensal bacteria make GPCR ligands that mimic human signalling molecules. Nature 549, 48–53 (2017).
Turpin-Nolan, S. M. et al. CerS1-derived C18:0 ceramide in skeletal muscle promotes obesity-induced insulin resistance. Cell Rep. 26, 1–10 e17 (2019).
Velagapudi, V. R. et al. The gut microbiota modulates host energy and lipid metabolism in mice. J. Lipid Res. 51, 1101–1112 (2010).
Caesar, R., Nygren, H., Oresic, M. & Backhed, F. Interaction between dietary lipids and gut microbiota regulates hepatic cholesterol metabolism. J. Lipid Res. 57, 474–481 (2016).
Moriya, T., Satomi, Y., Murata, S., Sawada, H. & Kobayashi, H. Effect of gut microbiota on host whole metabolome. Metabolomics 13, 101 (2017).
Chakravarthy, M. V. et al. “New” hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis. Cell Metab. 1, 309–322 (2005).
Pedersen, H. K. et al. Human gut microbes impact host serum metabolome and insulin sensitivity. Nature 535, 376–381 (2016).
Sun, L. et al. Gut microbiota and intestinal FXR mediate the clinical benefits of metformin. Nat. Med. 24, 1919–1929 (2018).
Gu, Y. et al. Analyses of gut microbiota and plasma bile acids enable stratification of patients for antidiabetic treatment. Nat. Commun. 8, 1785 (2017).
Karlsson, F. H. et al. Gut metagenome in European women with normal, impaired and diabetic glucose control. Nature 498, 99–103 (2013).
Le Chatelier, E. et al. Richness of human gut microbiome correlates with metabolic markers. Nature 500, 541–546 (2013).
Forslund, K. et al. Disentangling type 2 diabetes and metformin treatment signatures in the human gut microbiota. Nature 528, 262–266 (2015).
Gonzalez, F. J., Jiang, C. & Patterson, A. D. An intestinal microbiota-farnesoid X receptor axis modulates metabolic disease. Gastroenterology 151, 845–859 (2016).
Tilg, H., Zmora, N., Adolph, T. E. & Elinav, E. The intestinal microbiota fuelling metabolic inflammation. Nat. Rev. Immunol. 20, 40–54 (2020).
Canfora, E. E., Jocken, J. W. & Blaak, E. E. Short-chain fatty acids in control of body weight and insulin sensitivity. Nat. Rev. Endocrinol. 11, 577–591 (2015).
Donia, M. S. & Fischbach, M. A. Small molecules from the human microbiota. Science 349, 1254766 (2015).
Koh, A., De Vadder, F., Kovatcheva-Datchary, P. & Backhed, F. From dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites. Cell 165, 1332–1345 (2016).
Schroeder, B. O. & Backhed, F. Signals from the gut microbiota to distant organs in physiology and disease. Nat. Med. 22, 1079–1089 (2016).
Szabo, G. Gut-liver axis in alcoholic liver disease. Gastroenterology 148, 30–36 (2015).
Bennett, B. J. et al. Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation. Cell Metab. 17, 49–60 (2013).
Agus, A., Planchais, J. & Sokol, H. Gut microbiota regulation of tryptophan metabolism in health and disease. Cell Host Microbe 23, 716–724 (2018).
Varel, V. H. & Bryant, M. P. Nutritional features of Bacteroides fragilis subsp. fragilis. Appl. Microbiol. 28, 251–257 (1974).
Koropatkin, N. M., Martens, E. C., Gordon, J. I. & Smith, T. J. Starch catabolism by a prominent human gut symbiont is directed by the recognition of amylose helices. Structure 16, 1105–1115 (2008).
Elhenawy, W., Debelyy, M. O. & Feldman, M. F. Preferential packing of acidic glycosidases and proteases into Bacteroides outer membrane vesicles. mBio 5, e00909–00914 (2014).
Bligh, E. G. & Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911–917 (1959).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Nueda, M. J., Tarazona, S. & Conesa, A. Next maSigPro: updating maSigPro bioconductor package for RNA-seq time series. Bioinformatics 30, 2598–2602 (2014).
Folch, J., Lees, M. & Sloane Stanley, G. H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497–509 (1957).
Penno, A. et al. Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids. J. Biol. Chem. 285, 11178–11187 (2010).
Bui, H. H., Leohr, J. K. & Kuo, M. S. Analysis of sphingolipids in extracted human plasma using liquid chromatography electrospray ionization tandem mass spectrometry. Anal. Biochem. 423, 187–194 (2012).
Acknowledgements
We thank Hylde Zipoli, Liz Chang, Inge Hansen, and Richard Deckelbaum for advice on lipid chemistry, and Claudia Frick for comments on the manuscript. This work was supported by NIH Director’s New Innovator Award (DP2 OD007444 to R.E.L.) and the Max Planck Society.
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E.L.J., T.S.W., and R.E.L. conceived of the experiments. E.L.J., S.L.H., A.B., A.L.G., A.T.G., T.S.W., R.E.L. designed the experiments. E.L.J., S.L.H., B.I.K., J.L.W., and A.B. performed the experiments. E.L.J. analyzed the data. E.L.J. and R.E.L. wrote the manuscript with help from S.L.H. and T.S.W.
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Johnson, E.L., Heaver, S.L., Waters, J.L. et al. Sphingolipids produced by gut bacteria enter host metabolic pathways impacting ceramide levels. Nat Commun 11, 2471 (2020). https://doi.org/10.1038/s41467-020-16274-w
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DOI: https://doi.org/10.1038/s41467-020-16274-w
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