High-fat diet activates a PPAR-δ program to enhance intestinal stem cell function.

Abstract

We previously reported (Beyaz et al., 2016Beyaz S. Mana M.D. Roper J. Kedrin D. Saadatpour A. Hong S.J. Bauer-Rowe K.E. Xifaras M.E. Akkad A. Arias E. et al.High-fat diet enhances stemness and tumorigenicity of intestinal progenitors.Nature. 2016; 531: 53-58Crossref PubMed Scopus (345) Google Scholar) that a long-term (9–14 months) pro-obesity high-fat diet (HFD 60% kcal fat, Research Diets D12492) altered intestinal stem cell (ISC) and progenitor cell biology in a Peroxisome Proliferator-Activated Receptor (PPAR)-dependent manner. We furthermore proposed that fatty acids in the HFD stimulated a transcriptional program by inducing activity of lipid-sensing PPAR transcription factors in ISCs, which mediated enhanced stemness in this dietary regimen. This HFD is a frequently utilized diet-induced obesity (DIO) model (Cohen et al., 2014Cohen P. Levy J.D. Zhang Y. Frontini A. Kolodin D.P. Svensson K.J. Lo J.C. Zeng X. Ye L. Khandekar M.J. et al.Ablation of PRDM16 and beige adipose causes metabolic dysfunction and a subcutaneous to visceral fat switch.Cell. 2014; 156: 304-316Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar; Softic et al., 2019Softic S. Meyer J.G. Wang G.X. Gupta M.K. Batista T.M. Lauritzen H. Fujisaka S. Serra D. Herrero L. Willoughby J. et al.Dietary Sugars Alter Hepatic Fatty Acid Oxidation via Transcriptional and Post-translational Modifications of Mitochondrial Proteins.Cell Metab. 2019; 30: 735-753 e734Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Recently, Li et al. raised concerns about the use of “chow diet” as a control diet in stem cell studies (Li et al., 2020Li W. Houston M. Peregrina K. Ye K. Augenlicht L.H. Effects of Diet Choice on Stem Cell Function Necessitate Clarity in Selection and Reporting.Cell Stem Cell. 2020; 27: 11-12Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). Chow diet is a widely used control diet in DIO studies (Cohen et al., 2014Cohen P. Levy J.D. Zhang Y. Frontini A. Kolodin D.P. Svensson K.J. Lo J.C. Zeng X. Ye L. Khandekar M.J. et al.Ablation of PRDM16 and beige adipose causes metabolic dysfunction and a subcutaneous to visceral fat switch.Cell. 2014; 156: 304-316Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar; Softic et al., 2019Softic S. Meyer J.G. Wang G.X. Gupta M.K. Batista T.M. Lauritzen H. Fujisaka S. Serra D. Herrero L. Willoughby J. et al.Dietary Sugars Alter Hepatic Fatty Acid Oxidation via Transcriptional and Post-translational Modifications of Mitochondrial Proteins.Cell Metab. 2019; 30: 735-753 e734Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), including those studying stem cell function in homeostasis and tumorigenesis (Fu et al., 2019Fu T. Coulter S. Yoshihara E. Oh T.G. Fang S. Cayabyab F. Zhu Q. Zhang T. Leblanc M. Liu S. et al.FXR Regulates Intestinal Cancer Stem Cell Proliferation.Cell. 2019; 176: 1098-1112 e1018Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), given its wide availability and lower cost compared to purified control diets, a significant concern during longer term (9–14 months) DIO studies. To assess whether using chow diet in DIO studies may have confounding effects as suggested in Li et al., we repeated key functional and mechanistic experiments reported in our previous study using matched purified control diet (HFC, Research Diets D12450J) and HFD (Research Diets D12492). We found that long-term HFD feeding elevated ISC numbers, enhanced organoid-forming capacity, and induced the expression of PPAR target genes in ISCs compared to HFC (Figures S1A–S1G), confirming observations when the control group was fed chow. Importantly, the gene expression analysis by Li et al. also confirmed our observation that the PPAR-δ signature genes (Fabp1, Cpt1a, and Hmgcs2) are consistently upregulated in response to HFD irrespective of whether chow or HFC was used as the comparison group. Li et al. studied descriptive differences in gene expression programs of ISCs in response to HFD when either chow or a purified diet was used as a control. Consistent with our previous data, their Gene Set Enrichment Analysis (GSEA) identified the PPAR pathway as the top functional pathway enriched for both HFD versus chow and HFD versus purified control diet comparisons. However, while Li et al. conclude that different control diets create discordant results in terms of HFD-induced upregulation of Ppard expression, that conclusion is based on comparing our qRT-PCR data to their RNA-seq data. In our previous paper, we used independent approaches to assess the expression levels of PPAR-δ and its target genes in ISCs and progenitors. While qRT-PCR revealed slight upregulation of Ppard expression in HFD ISCs but not in progenitors, RNA-seq and western blot analysis of both ISCs and progenitors showed no significant changes in mRNA and protein levels between HFD and controls. However, we consistently observed significant upregulation of PPAR-δ target gene expression at the mRNA and protein levels in HFD ISCs and progenitors using orthogonal approaches, including Fabp1, Cpt1a, and Hmgcs2 using gain- and loss-of function experiments (Beyaz et al., 2016Beyaz S. Mana M.D. Roper J. Kedrin D. Saadatpour A. Hong S.J. Bauer-Rowe K.E. Xifaras M.E. Akkad A. Arias E. et al.High-fat diet enhances stemness and tumorigenicity of intestinal progenitors.Nature. 2016; 531: 53-58Crossref PubMed Scopus (345) Google Scholar) (Figure S1H). PPAR-δ is a ligand-induced transcription factor and its expression levels need not imply functional activity (Beyaz and Yilmaz, 2016Beyaz S. Yilmaz O.H. Molecular Pathways: Dietary Regulation of Stemness and Tumor Initiation by the PPAR-δ Pathway.Clin. Cancer Res. 2016; 22: 5636-5641Crossref PubMed Scopus (21) Google Scholar). Hence, our previous paper focused not on the expression levels of PPAR-δ but the fatty-acid-induced activation of a PPAR-δ program in regulating ISC function and tumorigenesis. To further substantiate the significance of PPAR-δ activation in regulating intestinal stemness, we have generated two new genetic models that enable inducible expression of a transcriptionally active form of PPAR-δ specifically in the intestinal epithelium (Villin-CreERT2-VP16) and ISCs (Lgr5-CreERT2-VP16), respectively (Kim et al., 2013Kim T. Zhelyabovska O. Liu J. Yang Q. Generation of an inducible, cardiomyocyte-specific transgenic mouse model with PPAR β/δ overexpression.Methods Mol. Biol. 2013; 952: 57-65Crossref PubMed Scopus (8) Google Scholar). We found that active PPAR-δ boosts ISC frequency and organoid formation and drives upregulation of PPAR-δ signature genes in intestinal crypts and ISCs, highlighting the fact that genetically enforced PPAR-δ signaling recapitulates the HFD response in ISCs (Figures S1I–S1L). Collectively, these data confirm the conclusions of our previous study. We also note several important variables that confound the comparisons performed by Li et al. First, in their study, the chow diet cohort consisted only of male mice and the HFD cohort consisted only of female mice (Figure S1M). Extensive experimental evidence and the National Institutes of Health (NIH) guidelines implicate sex as a key biological variable. Sex differences in the molecular and metabolic effects of DIO are well established, especially in the age of mice and diet duration studied by Li et al. (Salinero et al., 2018Salinero A.E. Anderson B.M. Zuloaga K.L. Sex differences in the metabolic effects of diet-induced obesity vary by age of onset.Int. J. Obes. 2018; 42: 1088-1091Crossref Scopus (25) Google Scholar), confounding interpretations of their gene expression results. Second, they performed their analysis after 3 months of HFD, whereas our published study used a much longer (>9 months) duration. Sex, age, and duration of the dietary regimens are extremely important for interpreting descriptive, functional, or mechanistic aspects of how diets influence ISCs. These key differences call into question the claims, justifications, and comparisons that Li et al. make regarding our published data (Beyaz et al.). Nevertheless, Li et al. confirmed our findings that an HFD robustly upregulates PPAR-δ signature genes (Fabp1, Cpt1a, and Hmgcs2) in ISCs irrespective of these confounding factors and issues with their experimental design and analysis discussed above, accentuating the reproducibility and robustness of our proposed mechanism. We support Li et al. in their call for clarity in selection and reporting of diets in stem cell research. However, as indicated above, conclusions about refuting effects of diet should be grounded in cohorts that are appropriately sex-matched and reproduce long-term feeding regimens and ages of mice used in original studies. Nonetheless, studying the effect of human nutrition on stem cell biology is difficult due to several variables including age, sex, diet composition, and population genetics. Yet, since our publication, causal mechanisms linking HFD and dietary lipids to stem cell function and cancer have been studied and our conclusions are supported in numerous publications (Wang et al., 2019Wang D. Fu L. Wei J. Xiong Y. DuBois R.N. PPARδ Mediates the Effect of Dietary Fat in Promoting Colorectal Cancer Metastasis.Cancer Res. 2019; 79: 4480-4490Crossref PubMed Scopus (17) Google Scholar; Zuo et al., 2019Zuo X. Deguchi Y. Xu W. Liu Y. Li H.S. Wei D. Tian R. Chen W. Xu M. Yang Y. et al.PPARD and Interferon Gamma Promote Transformation of Gastric Progenitor Cells and Tumorigenesis in Mice.Gastroenterology. 2019; 157: 163-178Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Mimicking diverse human diets and their impact on intestinal tumorigenesis is challenging; however, every model provides important mechanistic clues. We believe that uncovering causal mechanisms by using diverse experimental approaches that range from in vitro organoids to in vivo models will greatly improve our understanding of how diet and nutrients influence stem cells in human health and disease. This work was supported by the National Institutes of Health ( P30CA045508-33 to S.B. and R01 CA211184 , CA254314 , DK126545 , U01CA250554 , and P30CA14051-43 to Ö.H.Y.), D.O.D. ( CA190699 to Ö.H.Y.), The Oliver S. and Jennie R. Donaldson Charitable Trust (S.B.), the STARR Cancer Consortium ( I13-0052 , S.B.), The MIT Stem Cell Initiative through Foundation MIT (Ö.H.Y.), the Pew Foundation (Ö.H.Y.) and a Koch Frontier Award (Ö.H.Y.). Conceptualization, S.B., M.D.M., and Ö.H.Y.; Investigation, S.B. and M.D.M.; Writing – Original Draft, S.B. with help from Ö.H.Y.; Writing – Review & Editing, S.B., M.D.M., and Ö.H.Y.; Visualization, S.B.; Supervision, S.B. and Ö.H.Y.; Funding Acquisition, S.B. and Ö.H.Y. Download .pdf (35.59 MB) Help with pdf files Document S1. Figure S1