Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we show that high-fat diet (HFD)-induced obesity augments the numbers and function of Lgr5+ intestinal stem cells of the mammalian intestine. Mechanistically, a HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-δ) signature in intestinal stem cells and progenitor cells (non-intestinal stem cells), and pharmacological activation of PPAR-δ recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-δ-dependent manner. Notably, HFD- and agonist-activated PPAR-δ signalling endow organoid-initiating capacity to progenitors, and enforced PPAR-δ signalling permits these progenitors to form in vivo tumours after loss of the tumour suppressor Apc. These findings highlight how diet-modulated PPAR-δ activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumours.
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This work was supported by the Howard Hughes Medical Institute (S.H.O. and D.M.S.), Ellison Medical Foundation Aging grant (D.M.S.), NIH (R01 CA103866 and AI47389; D.M.S.), NIH (K08 CA198002; J.R.), Department of Defense PRCRP Career Development Award CA120198 (J.R.), NIH (R00 AG045144; Ö.H.Y.), NIH (R00 AG041765; D.W.L.), Center for the Study of Inflammatory Bowel Diseases from the Massachusetts General Hospital NIH (DK043351; Ö.H.Y.), NIH Cancer Center Support (core) grant P30-CA14051 (Ö.H.Y.), Kathy and Curt Marble Cancer Research Fund (Ö.H.Y.), American Federation of Aging Research (AFAR; Ö.H.Y.), and V Foundation Scholar grant (J.R. and Ö.H.Y.). M.D.M. is supported by a Koch MIT Ludwig Center post-doctoral fellowship, D.K. receives fellowship support from MGH (T32DK007191), and M.M.M. is a Robert Black Fellow of the Damon Runyon Cancer Research Foundation. We thank the Koch Institute Swanson Biotechnology Center (SBC) for technical support, specifically the Hope Babette Tang (1983) Histology Facility and Kathleen Cormier. We thank S. Holder for superior histology and help with special stains. We thank P. Wisniewski and G. Paradis of the Whitehead flow cytometry and Koch core facilities, respectively, for their expertise in cell sorting. We thank members of the Yilmaz laboratory for discussions.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 A HFD alters intestinal morphology and enhances intestinal progenitor proliferation.
a–g, In comparison to mice fed a standard chow, mice on a HFD gained on average 50% mass (a, control: n = 11, HFD: n = 15), had reduced small intestinal mass and length (b, c, control: n = 11, HFD: n = 15), longer crypts and shorter villi (e, g, n = 3 each), and fewer villus enterocytes (f, n = 3). HFD did not change the density of crypts (d, n = 3) in the proximal jejunum. The proximal jejunum was defined as the length between 6 and 9 cm as measured from the pylorus (the distal portion of the stomach). h–k, HFD enhanced BrdU incorporation in ISCs (or crypt base columnar cells) and progenitor cells (or transit-amplifying cells) in the proximal jejunum (h, n = 6) and sorted cell populations (i, n = 3) after a 4-h pulse. HFD increased the total (j, control: n = 4, HFD: n = 5) and normalized numbers of BrdU-labelled enterocytes compared to controls after a 24-h pulse. Arrowhead (k) marks the leading edge of migrating BrdU-positive enterocyte. l, m, Representative images of Olfm4 (l, n = 3) and Crp4 (m, n = 6) in situ hybridizations from control and HFD-fed mice. n, No significant difference in the number of jejunal caspase3+ cells was detected by immunohistochemistry. Images are representative of three separate experiments (n = 3); arrowheads indicate representative caspase3+ enterocytes. o, The HFD chow (Research Diets D12492) provides a higher percentage of kilocalories from fat and conversely a lower percentage of kilocalories from protein and carbohydrates compared to a standard chow diet (Labdiet RMH3000). Unless otherwise indicated, data are mean ± s.d. from n independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-tests). Scale bars, 100 μm (g, k, n), 50 μm (h, i, k (inset), l, m) and 20 μm (l, m, insets); two separate fields of jejunum (d), and at least 15 crypts (e), 15 villi (f), 10 villi (g), 100 cells (i), 25 villi (j) and 25 crypt–villus units (n) were counted per sample in each independent experiment.
Extended Data Figure 2 A HFD and PPAR-δ signalling have minimal effects on enteroendocrine and goblet cell differentiation but promote intestinal regeneration after 15 Gy irradiation.
a, b, Quantification (a, n = 3) of immunostains (b, n = 3) for chromogranin A revealed no difference in the numbers of jejunal enteroendocrine cells (arrowheads) per crypt–villus unit in HFD-fed mice and GW501516-treated mice compared to their respective controls. c, d, Quantification (c, n = 4) of Alcian blue/PAS staining (d, n = 4) showed no difference in mucinous goblet cells (arrowhead) in HFD-fed and GW501516-treated mice compared to their respective controls. e, f, A HFD increased the number of regenerating crypts as measured by an increased number of crypts containing at least ten Ki67+ (a marker of proliferation) cells (e, n = 3) or at least one Olfm4+ cell (f, n = 3) per 5 mm of jejunum by immunohistochemistry (IHC) or in situ hybridization (ISH). Arrows indicate Olfm4+ crypts. g, Surviving crypt numbers after ionizing irradiation-induced (XRT) damage. Arrows denote regenerating crypts; asterisks denote aborted crypts (n = 3). Unless otherwise indicated, data are mean ± s.d. from n independent experiments. Scale bars, 100 μm (b, d), 50 μm (e–g) and 20 μm (e, f, insets); 50 crypt–villus units per sample were analysed (a, c) and approximately 50 crypts (e–g) were counted per sample in each independent experiment.
Extended Data Figure 3 A HFD and fatty acids do not activate inflammatory pathways in intestinal crypts and organoids, while HFD and enforced PPAR-δ signalling enhance colonic stem-cell function.
a, A HFD did not alter the normalized expression levels of inflammatory genes from the GSEA Molecular Signature Database (MSigDB; signature M6557) data set in ISCs and progenitors. b, A HFD did not induce differential expression of ‘inflammatory response’ genes from Gene Ontology (GO; 0006954) in ISCs (Lgr5-GFPhi) or progenitors (Lgr5-GFPlow) compared to control. Fold changes of GO inflammatory response genes are indicated in red, and fold changes for all other genes are indicated in blue. c, HFD did not activate the NF-κB or the STAT-3 pathways in the intestinal crypt. Total and phosphorylated protein levels in crypt lysates were assessed by immunoblots (n = 3). For western blot source data, see Supplementary Fig. 1. d, A HFD did not induce pro-inflammatory gene expression in ISCs (Lgr5-GFPhi) or progenitors (Lgr5-GFPlow). Relative expression levels compared to Actb were measured by qRT–PCR (n = 5). e, Ex vivo palmitic acid, lipid mixture or GW501516 treatment did not induce inflammatory gene expression in crypt-derived organoids compared to vehicle. Relative expression levels compared to Actb were assessed by qRT–PCR (n = 4, 12 wells per sample were analysed). f, A HFD boosted the number of BrdU-labelled cells as measured in distal colonic crypts compared to control (control n = 6, HFD n = 5) after a 4-h pulse. g, A HFD increased the frequency of colonic ISCs (Lgr5-GFPhi, dark green) and progenitor cells (Lgr5-GFPlow, light green) (n = 8). h, i, A HFD enhanced PPAR-δ (h) and β-catenin (i) target gene expression in colonic ISCs and progenitors. Relative expression levels compared to Actb were determined by qRT–PCR (n = 5, all fold changes are significant with P < 0.05). j–m, Colonic crypts derived from HFD-fed (j, n = 4; k, n = 4) and GW501516-treated (l, n = 5; m, n = 4) mice demonstrated greater primary and secondary organoid-forming capacity compared to their respective controls. Representative images: day-4 organoids. Unless otherwise indicated, data are mean ± s.d. from n independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-tests). Scale bars, 50 μm (f), 100 μm (j) and 200 μm (l); 50 crypts per sample were analysed (f) in each independent experiment.
a, HFD organoids contained higher frequencies of ISCs (Lgr5-GFPhi) compared to control (n = 3). b, c, Control and HFD organoids demonstrated no differences in morphologic ultrastructure as seen in 1-μm sections of control (left) and HFD (right) organoids counterstained with Toluidine Blue (b), and electron microscopy images of representative control (left) and HFD (right) organoids (c) (n = 3). d, e, Composition of organoids derived from control (d) and HFD (e) crypts as assessed by single-cell gene expression analysis. Organoids on day 5 contained ISCs (Lgr5 and Olfm4), Paneth cells (Lyz), enteroendocrine cells (Chga), and goblet cells (Muc2). Forty-eight live cells per group were sorted and single-cell gene expression analysis was performed after pre-amplification using corresponding stem-cell and lineage primers (see Methods). f, Crypt-derived organoids from control or HFD-fed mice included chromogranin A-, mucin 2- and lysozyme-positive cells as assessed by immunofluorescence (blue = DAPI, red = cell-specific antibody). Images represent two experiments (n = 2). g, Cultured villi from control and HFD-fed mice lack the ability to form organoids. Images represent two experiments with 6 wells per sample (n = 2). h, ISCs from HFD-fed mice contained greater organoid-forming potential compared to controls. Arrowheads indicate representative organoids at days 4, 7 and 10 of culture (n = 4). i, Individually dissociated HFD primary organoids that were derived from single ISCs possessed more secondary organoid-forming ability than those from controls. (n = 4). Representative images: day-4 secondary organoids. j, k, Single-cell gene expression analysis revealed that ISCs from both control (j) and HFD (k) mice can beget Paneth cells (Lyz) within 24 h in culture (48 cells per group, see Methods). l, m, Composition of organoids derived from control (l) and HFD (m) ISCs (Lgr5-GFPhi) as assessed by single-cell gene expression analysis (48 cells per group, see Methods). Organoids on day 5 contained ISCs (Lgr5 and Olfm4), Paneth cells (Lyz), endocrine cells (Chga) and goblet cells (Muc2). Unless otherwise indicated, data are mean ± s.d. from n independent experiments; *P < 0.05 (Student’s t-tests). Scale bars, 20 μm (b), 2 μm (c), 50 μm (f), 200 μm (g, i) and 100 μm (h).
Extended Data Figure 5 Ex vivo exposure of mouse and human organoids to fatty acids recapitulates aspects of a HFD.
a, b, Individually dissociated primary organoids possessed more secondary organoid-forming activity (a, n = 4, the mean number of secondary organoids subcloned from each of 5 primary organoids in 4 independent experiments), and contained a higher frequency of Lgr5-GFPhi ISCs (b, n = 3) after 4 weeks of treatment with 30 μM palmitic acid compared to vehicle. c, Exposure of naive crypts to 30 μM oleic acid had no effect on primary organoid formation measured at day 7 (n = 6). Representative images: day-7 organoids. d, Individually dissociated primary organoids possessed more secondary-organoid-forming capacity after 4 weeks of treatment with 30 μM oleic acid (n = 4, the mean number of secondary organoids subcloned from each of 5 primary organoids in 4 independent experiments) compared to vehicle (same vehicle cohort used in a and d). e, Lipid mixture composition (Sigma L0288) as described by the manufacturer. f, Ex vivo treatment of human-derived small intestinal crypts (H1–H4) passaged in the presence of lipid mixture, palmitic acid or GW501516 augmented relative clonogenicity compared to vehicle, as shown in representative images from 4 independent experiments. H1: n = 10 (vehicle, palmitic acid, GW501516) and n = 6 (lipid mixture) wells were analysed. H2: n = 16 (vehicle), n = 6 (lipid), n = 12 (palmitic acid) and n = 14 (GW501516) wells were analysed. H3: n = 10 (vehicle), n = 12 (lipid, palmitic acid) and n = 8 (GW501516) wells were analysed. H4: n = 7 (vehicle, GW501516), n = 6 (lipid) and n = 9 (palmitic acid) wells were analysed. Age, gender and BMI are specified. g–j, Human crypt-derived organoids after ex vivo treatment with palmitic acid, lipid or GW501516 induced PPAR-δ target gene expression as assessed in passaged cultures with qRT–PCR (n = 4, 12 wells per sample were analysed, all fold changes are significant, P < 0.05). Unless otherwise indicated, data are mean ± s.d. from n independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-tests). Scale bars, 100 μm (c) and 500 μm (f).
Extended Data Figure 6 PPAR-δ is the predominant PPAR family member expressed in intestinal progenitors and mediates the effects of HFD.
a, PPAR-δ is the most abundant PPAR family member in ISCs (Lgr5-GFPhi) and progenitors (Lgr5-GFPlow) based on RNA-seq data. b, Confirmation of PPAR family member mRNA expression levels in ISCs (Lgr5-GFPhi) and progenitors (Lgr5-GFPlow) by qRT–PCR (n = 5). c, Genes upregulated in HFD ISCs (Lgr5-GFPhi) versus control ISCs were enriched in PPAR and LXR/RXR motifs. d, GSEA of RNA-seq data identified enrichment of PPAR-δ targets in ISCs (Lgr5-GFPhi) and progenitors (Lgr5-GFPlow) with a HFD. e, Confirmation of induced PPAR-δ target gene expression in flow-sorted ISCs (Lgr5-GFPhi) and progenitors (Lgr5-GFPlow) by qRT–PCR (n = 5). All fold changes were significant, P < 0.05. f, g, Representative images of Olfm4+ (ISCs, f) and Crp4+ (Paneth cells, g) in situ hybridization from vehicle and GW501516-treated mice (f, n = 3; g, n = 4). h, Ex vivo exposure of organoids to palmitic acid, lipid mixture or GW501516 stimulated PPAR-δ and β-catenin target gene expression (n = 3, all fold changes were significant, P < 0.05). i, j, Injection with tamoxifen (4 injections on alternating days) in PpardL/L; Villin-CreERT2 mice led to efficient intestinal deletion (IKO) of Ppard (7 days after the last tamoxifen dose), as assessed by allele-specific deletion PCR (i, n = 3) and immunoblot analysis (j, n = 3) of crypts. For western blot source data, see Supplementary Fig. 1. k, Acute disruption of Ppard (8 days after the last tamoxifen dose) did not perturb ISC and progenitor proliferation, as determined 4-h after BrdU administration (n = 3). l, m, Acute Ppard deletion (8 days after the last tamoxifen dose) did not significantly alter Olfm4+ ISCs numbers (L/L: n = 5, IKO: n = 4) (l) or Crp4+ Paneth cell (n = 5) (m) numbers, as assessed by in situ hybridization. n, Loss of Ppard transcripts in Ppard IKO organoids was confirmed by qRT–PCR using deletion-specific primers (n = 3). o, PPAR-δ is required for the induction of PPAR-δ and β-catenin target gene expression in secondary organoids after ex vivo palmitic acid, lipid or GW501516 treatment (n = 5, all fold changes are significant, P < 0.05). p, Heat map of differentially expressed genes illustrated induction of a PPAR-δ program in HFD-derived ISCs and progenitors relative to controls. Unless otherwise indicated, data are mean ± s.d. from n independent experiments; *P < 0.05 (Student’s t-tests). Scale bars, 50 μm (f, g, k–m) and 20 μm (insets); 50 crypts per sample were analysed in each independent experiment (f, g, k–m).
Extended Data Figure 7 HFD and PPAR-δ signalling boost nuclear β-catenin localization and activity in intestinal progenitors.
a, b, HFD-derived ISCs (a, Lgr5-GFPhi) and progenitors (b, Lgr5-GFPlow) required less Wnt3a and R-spondin to initiate organoids than control ISCs, as measured by comparing organoid-formation in complete ENRW media, which includes EGF, Noggin, R-spondin and Wnt3a, versus EN media, which includes EGF and Noggin but lacks Wnt3a and R-spondin (n = 3). Control-derived progenitors, in contrast to HFD-derived progenitors, rarely formed organoids in either ENRW or EN media. c–f, HFD increased nuclear β-catenin localization in flow-sorted ISCs and progenitors from HFD (c, n = 5) and GW501516-treated (d, n = 4) mice as determined by immunofluorescence (red, DAPI; cyan, non-phosphorylated β-catenin, CST 8814S). At least 100 cells per sample were quantified. Representative images are shown in e and f. g–i, HFD (g) and GW501516 (h) treatment increased the numbers of ISCs and progenitors with β-catenin+ nuclei, as assessed by immunostaining (n = 4 each). Representative images are shown in i; arrowheads indicate representative nuclear β-catenin in ISCs (red) and progenitors (black). j–l, Association of PPAR-δ and β-catenin in control and HFD-derived intestinal crypts as shown by immunoprecipitation (IP) (n = 3). For western blot source data, see Supplementary Fig. 1. Unless otherwise indicated, data are mean ± s.d. from n independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-tests). Scale bars, 50 μm (e, f) and 20 μm (i); organoid assays: 2–4 wells per sample analysed (a, b), 50 crypts per sample were analysed in each independent experiment (g, h).
Extended Data Figure 8 HFD-mediated alterations in β-catenin target gene expression in single ISCs and progenitors.
a, Heat map representation of β-catenin target gene expression in single ISCs (Lgr5-GFPhi, 24 cells) and progenitors (Lgr5-GFPlow, 72 cells) (see Methods). b, Stem-cell signature genes were identified by comparing target gene expression in control ISCs (Lgr5-GFPhi) to control progenitors (Lgr5-GFPlow). c, HFD signature genes were identified by comparing target gene expression in HFD ISCs to control ISCs (Lgr5-GFPhi). d, t-Distributed stochastic neighbour embedding (tSNE) analysis of single cells using all β-catenin target genes. e, tSNE analysis of single cells using stem-cell signature genes. f, tSNE analysis of single cells using HFD signature genes. g, h, Lgr5 expression was similar in HFD ISCs (Lgr5-GFPhi) (g) and progenitors (Lgr5-GFPlow) (h) as compared to their respective controls. i, j, HFD increased the percentage of ISCs (Lgr5-GFPhi) (i) and progenitors (Lgr5-GFPlow) (j), with increased Jag1 and Jag2 expression compared to their respective controls. k, l, HFD (k, n = 3) and GW501516 treatment (l, n = 4) augmented Jag1 expression compared to control and vehicle treatments, respectively, as assayed by single-molecule in situ hybridization: Jag1 is broadly expressed throughout the crypt. Unless otherwise indicated, data are mean ± s.d. from n independent experiments; *P < 0.05 (Student’s t-tests). Scale bars, 50 μm (k, l) and 20 μm (insets); more than 50 crypts per sample were analysed in each independent experiment (k, l). See Supplementary Information for raw gene expression data.
a–f, At 4–5 months of age, homozygous db/db mice gained on average 50% more mass (a, n = 9), had increased small intestinal mass and length (b, c, n = 9), shallower crypts (d, n = 4) and longer villi (e, f, n = 5) than control db/+ mice. g, h, Immunostains for OLFM4 (n = 6) and lysozyme (n = 6) revealed a slight reduction in the number of Olfm4+ ISCs and Paneth cells, respectively, in db/db mice compared to db/+ controls. i, Organoid-forming capacity of db/db crypts was higher (P = 0.095) than db/+ controls (n = 7). j, Single-cell gene expression analysis revealed no induction of PPAR-δ or β-catenin target gene expression in live, enriched stem and progenitor cells that are depleted of secretory cells (7-AAD−Epcam+CD24−c-kit− cells, 48 cells per group; see Methods) from db/db intestines compared to control intestines. Unless otherwise indicated, data are mean ± s.d. from n independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-tests). Scale bars, 100 μm (f), 50 μm (g, h), 20 μm (g, h, insets) and 200 μm (i); and at least 30 crypts (d), 20 villi (e) and 100 crypts (g, h) were assessed per sample in each independent experiment. All db/db and db/+ mice were fed a standard chow diet.
Extended Data Figure 10 PPAR-δ activation bestows adenoma-initiating capacity to Apc-null progenitors.
a, b, Representative optical endoscopy images (top) from Fig. 5, with H&E (middle) and β-catenin (immunohistochemistry, bottom) sections of adenomas derived from orthotopic transplantation of Apc-null ISCs (a, Lgr5-GFPhi) and progenitors (b, Lgr5-GFPlow) from vehicle- and GW501516-treated mice 4 days after Apc deletion. Tumours exhibited hyperchromasia, lack of maturation, nuclear crowding and nuclear β-catenin positivity. Two independent pathologists blinded to treatment groups interpreted the results. c, d, Apc deletion was confirmed in sorted small intestinal ISCs and progenitors from vehicle- and GW501516-treated ApcL/L; Lgr5-EGFP-IRES-CreERT2 mice 4 days after tamoxifen administration (c, n = 3) and in isolated tumours (d, n = 3) by PCR amplification, using allele-specific deletion primers targeting exon 14. Unless otherwise indicated, n represents independent experiments. Scale bars (a, b), 50 μm (20×) and 20 μm (60×).
This file comprises: Supplementary Figure 1, which shows the original uncropped images of western blots presented in Figure 3 and in Extended Data Figures 3, 6 and 7; Supplementary Notes, which include texts for clarifying analyses and discussion; and Supplementary Tables 1 – 2, which show the lists of primers used in qRT-PCR and single cell analysis respectively. (PDF 3411 kb)
This file contains the raw gene expression data for the single cell gene expression analyses. (XLSX 238 kb)
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Beyaz, S., Mana, M., Roper, J. et al. High-fat diet enhances stemness and tumorigenicity of intestinal progenitors. Nature 531, 53–58 (2016). https://doi.org/10.1038/nature17173
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