International Journal of Obesity (2014),1–9 © 2014 Macmillan Publishers Limited All rights reserved 0307-0565/14 www.nature.com/ijo ORIGINAL ARTICLE Impact of high-fat feeding on basic helix–loop–helix transcription factors controlling enteroendocrine cell differentiation This article has been corrected since online publication and a corrigendum appears in this issue Y Sakar1,2, FA Duca1,2, B Langelier1,2, F Devime1,2, H Blottiere1,2, C Delorme1,2, P Renault1,2 and M Covasa1,2,3,4 BACKGROUND AND OBJECTIVES: Gut hormones secreted by enteroendocrine cells (EECs) play a major role in energy regulation. Differentiation of EEC is controlled by the expression of basic helix–loop–helix (bHLH) transcription factors. High-fat (HF) feeding alters gut hormone levels; however, the impact of HF feeding on bHLH transcription factors in mediating EEC differentiation and subsequent gut hormone secretion and expression is not known. METHODS: Outbred Sprague–Dawley rats were maintained on chow or HF diet for 12 weeks. Gene and protein expression of intestinal bHLH transcription factors, combined with immunofluorescence studies, were analyzed for both groups in the small intestine and colon. Gut permeability, intestinal lipid and carbohydrate transporters as well as circulating levels and intestinal protein expression of gut peptides were determined. RESULTS: We showed that HF feeding resulted in hyperphagia and increased adiposity. HF-fed animals exhibited decreased expression of bHLH transcription factors controlling EEC differentiation (MATH1, NGN3, NEUROD1) and increased expression of bHLH factors modulating enterocyte expression. Furthermore, HF-fed animals had decreased number of total EECs and L-cells. This was accompanied by increased gut permeability and expression of lipid and carbohydrate transporters, and a decrease in circulating and intestinal gut hormone levels. CONCLUSIONS: Taken together, our results demonstrate that HF feeding caused decreased secretory lineage (that is, EECs) differentiation through downregulation of bHLH transcription factors, resulting in reduced EEC number and gut hormone levels. Thus, impaired EEC differentiation pathways by HF feeding may promote hyperphagia and subsequent obesity. International Journal of Obesity advance online publicatin, 25 February 2014; doi:10.1038/ijo.2014.20 Keywords: intestinal chemosensation; enteroendocrine cells; gut peptides; GLP-1 INTRODUCTION in Notch signaling in the intestine downregulate the absorptive The gastrointestinal tract constitutes the largest endocrine organ enterocyte lineage and increase cell numbers of the secretory 10 in the body.1 Enteroendocrine cells (EECs) secrete a variety of gut lineage. Three specific Notch-regulated basic helix–loop–helix hormones in response to intestinal nutrients, which play a major (bHLH) transcription factors are sequentially involved in the role in the control of food intake and regulation of energy different stages of secretory and absorptive cell differentiation: homeostasis.2,3 Obesity is associated with altered gut hormone MATH1, neurogenin 3 (NGN3) and neurogenic differentiation 1 11 levels, and therefore EECs have been proposed as possible targets (NEUROD1). for treatment of this disease.4 The intestinal epithelium is rapidly The proneural transcription factor mouse atonal homolog 1 regenerating and proliferating. This constant turnover is governed (also known as MATH1) is present in the GI tract throughout by an active pluripotent intestinal stem cell population, giving rise development and has been localized in intestinal epithelial villi 12 to five types of epithelial cells, which are represented by an and immature crypts. MATH1 null mice lack three secretory cell 12 absorptive enterocyte lineage or a secretory lineage composed of types: goblet, paneth and EECs. Transgenic MATH1 mice exhibit goblet cells, enteroendocrine cells, paneth cells and tuft cells.5,6 a large expansion in secretory cells and near-complete loss of Importantly, Notch and Wnt signaling pathways appear to play a absorptive cells, further indicating that MATH1 is a key transcrip- critical role in the fate between differentiation into the absorptive tion factor in directing intestinal progenitors to adopt the or secretory lineages.7,8 This effect is mediated through hairy/ secretory lineage.13 Second, NGN3, expressed in endocrine enhancer of split (HES1), a transcription factor whose expression is progenitor cells, is essential for the sequential differentiation of initiated by the Notch signaling pathway and is a repressor of cell secretory cells into endocrine lineage specification, as evidenced cycle inhibitors.9 Activation of Notch/HES1 signaling promotes by mice carrying a NGN3 null mutation failing to develop differentiation of the absorptive lineage and decreases differentia- intestinal endocrine cells.14,15 Although NGN3 is not expressed tion of the secretory lineage.8 Therefore, it follows that disruptions in mature endocrine cells, it is known to stimulate pro-endocrine 1UMR1913-MICALIS, INRA Centre de Recherche de Jouy-en-Josas, Jouy-en-Josas, France; 2UMR1913-MICALIS, AgroParisTech, Jouy-en-Josas, France; 3Department of Basic Medical Sciences, College of Osteopathic Medicine, Western University of Health Sciences, Pomona, CA, USA and 4Department of Human Health and Development, University of Suceava, Suceava, Romania. Correspondence: Dr M Covasa, UMR1913-MICALIS, INRA Centre de Recherche de Jouy-en-Josas, Domaine de Vilvert, Jouy-en-Josas Cedex 78350, France. E-mail: [email protected] Received 18 October 2013; revised 3 December 2013; accepted 17 January 2014; accepted article preview online 31 January 2014 Dietary fat and enteroendocrine cells Y Sakar et al 2 transcription factors, such as NEUROD1, paired box 4 and 6 (PAX4 reverse transcribed into 100 μl complementary DNA using the cDNA kit and PAX6), winged helix factors 1/2 (FOXA 1/2) and transcriptional (Applied Biosystem, Courtabeouf, France). Subsequent complementary regulatory factor 6 (RFX6).16–18 These and other pro-endocrine DNA was diluted fivefold and used for quantitative real-time PCR using Taqman gene expression master mix and inventoried Taqman gene transcription factors are thought to be responsible for differentia- 26 1 expression assays in an ABI Prism as previously described. Relative mRNA tion and maintenance of the mature endocrine cell populations. −ΔΔ expression was quantified using the 2 CT method with β-actin as For example, NEUROD1 regulates transcription of secretin and internal control. cholecystokinin (CCK), as NEUROD1 − / − mice lack secretin and CCK cells (S and I cells, respectively), although they maintain many other EECs, including glucagon-like peptide-expressing L-cells Western blotting (GLP-1, GLP-2).19 However, mice with a FOXA1/2 null mutation Isolated intestinal epithelial cell (n = 6 per group) aliquots were suspended have a reduced number of GLP-1 and polypeptide YY (PYY) in 500 ml of radioimmunoprecipitation assay buffer containing protease expressing L-cells via PAX6 downregulation.16 Additionally, the inhibitor cocktails (Sigma). Intestinal cells were lysed, homogenized and centrifuged for 30 min at 13 000 g at 4 °C. Soluble protein (50–100 μg) was transcription regulatory factor RFX6 plays a major role in K-cell- − – mediated gastric inhibitory polypeptide (GIP) expression,18 as run on SDS PAGE gels containing 8 12% bis/acrylamide, transferred to nitrocellulose membranes, and incubated with anti-GLP-1/2, PYY, CCK, deletion of RFX6 abolishes K-cell differentiation and GIP 20 secretin, GIP, NGN3, MATH1, PAX6, HES1, E74-like factor 3 (ELF3), CR6- expression. interacting factor 1 (CRIF1), SGLT1, glucose transporter 2/5 (GLUT2/5), fatty High-fat (HF) feeding is known to promote the development of acid transporter protein (FATP), Zonula-1 (ZO-1), phosphorylating myosin obesity, and is associated with reduction in circulating gut light chain (p-MLC) and occludin antibodies (Santa Cruz Biotechnology, hormone levels. Few studies have directly examined the effect Santa Cruz, CA, USA) and FOXA1/2 (Thermo Scientific-Pierce, Rockford, IL, of HF feeding on EEC changes, with conflicting results.21,22 Two USA). Immune complexes were detected by chemiluminescence (GE studies indicate no significant changes in EEC number after HF Healthcare, Saclay, France). Quantification was performed by scanning β feeding,22,23 while one recent study showed a significant increase densitometry using ImageJ (NIH, Bethesda, MD, USA) against -actin as in K- and L-cell number, in spite of reduced GLP-1 secretion.24 We internal control. have demonstrated that feeding a HF diet resulted in decrease in L cells of obese-prone rats and circulating GLP-1 levels.4 However, Immunofluorescence the mechanisms by which HF diet modulates EEC differentiation The small intestine (n = 4 per group) and colon (n = 5 per group) were fixed remain unclear. To our knowledge, only one study has examined and cut into 4-μm-thick sections using standard procedures.27 After the effect of HF feeding on EEC differentiation, demonstrating that deparaffinizing and rehydrating, slides were placed in 6% hydrogen diet-induced obesity is associated with increased RFX6 and, peroxide for 20 min, and blocked with phosphate-buffered saline/3% accordingly, increased GIP expression in enteroendocrine K-cells.25 bovine serum albumin/6% goat serum for 1 h. Sections were incubated Therefore, in the current study, we examined the effects of overnight at 4 °C with rabbit polyclonal antibody raised against MATH1 (1:100, Santa