Volume 155, Issue 2, Pages 501-513 (August 2018) Disruption of Epithelial HDAC3 in Intestine Prevents Diet-Induced Obesity in Mice  Jordan Whitt, Vivienne Woo, Patrick Lee, Jessica Moncivaiz, Yael Haberman, Lee Denson, Patrick Tso, Theresa Alenghat  Gastroenterology  Volume 155, Issue 2, Pages 501-513 (August 2018) DOI: 10.1053/j.gastro.2018.04.017 Copyright © 2018 AGA Institute Terms and Conditions

Gastroenterology 2018 155, 501-513DOI: (10.1053/j.gastro.2018.04.017) Copyright © 2018 AGA Institute Terms and Conditions

Figure 1 Intestinal epithelial HDAC3 expression regulates development of diet-induced obesity. (A) Correlation between HDAC3 expression in the ileum and body weight Z scores across 43 pediatric patients, confirmed with lack of intestinal inflammation by histology. (B) HDAC3 expression in patients from (A) based on body weight Z score stratification. (C) HDAC3 expression in IECs from HDAC3FF or HDAC3ΔIEC mice by real-time PCR. (D–G) Male, age-matched, C57BL/6 control (HDAC3FF) and HDAC3ΔIEC mice were fed (D, E) normal chow or an (F, G) HFD. (D, F) Body weight. (E, G) Body composition (% body fat and % lean tissue) quantified at 20 weeks. Data represent 4–8 mice per group. Results are shown as mean ± standard error of the mean. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. Gastroenterology 2018 155, 501-513DOI: (10.1053/j.gastro.2018.04.017) Copyright © 2018 AGA Institute Terms and Conditions

Figure 2 IEC-intrinsic deletion of HDAC3 improves obesity-associated glucose and lipid abnormalities. (A) Glucose tolerance test (GTT) and (B) serum insulin for HDAC3FF and HDAC3ΔIEC mice fed normal chow or HFD for 20 weeks. (C-E) Female, age matched, C57BL/6 control (HDAC3FF) and HDAC3ΔIEC mice fed HFD. (C) Body weight (D) % body fat and (E) GTT analyzed at 20 weeks HFD feeding. (F) Serum triglycerides and (G) H&E stained sections of liver (top) and adipose (bottom) in mice on HFD, scale bar 20μm. Data represent 4-8 mice per group. Results are shown as mean ± standard error of the mean. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. min, minute. Gastroenterology 2018 155, 501-513DOI: (10.1053/j.gastro.2018.04.017) Copyright © 2018 AGA Institute Terms and Conditions

Figure 3 HDAC3ΔIEC mice show increased energy expenditure. (A) Body weight of HDAC3FF and HDAC3ΔIEC male mice fed HFD for 16 weeks. (B) Food intake for individually housed mice in (A) measured over 24 hours in metabolic chambers. (C) Physical activity measured by photobeam breaks. (D) Heat generation, (E) CO2 production, and (F) O2 consumption. Data represent 4–6 mice per group. Results are shown as mean ± standard error of the mean. ∗∗P < .01, ∗∗∗P < .001. Gastroenterology 2018 155, 501-513DOI: (10.1053/j.gastro.2018.04.017) Copyright © 2018 AGA Institute Terms and Conditions

Figure 4 HDAC3 regulates expression of microbiota-dependent metabolic pathways in IECs. RNA sequencing was performed on IECs isolated from mid-distal small intestine. Volcano plots of differential gene expression between (A) germ-free (GF) vs microbiota-replete conventionally housed (CNV) mice and (B) HDAC3FF vs HDAC3ΔIEC mice, n = 2 per group. (C) Venn analysis of genes enriched in lipid metabolism that are dependent on the microbiota and HDAC3. Heat-map of relative transcript abundance of overlapping genes. (D) mRNA expression by real-time PCR in IECs from the small intestine. (E) ChIP–quantitative PCR comparing H3K9Ac levels at phosphoenolpyruvate carboxykinase 1 (PCK1) promoter in IECs. (F) TRANSFAC identification of transcription factor binding motifs within hyperacetylated region of PCK1 promoter. Data represent 3–6 mice per group. Results are shown as mean ± standard error of the mean. ∗P < .05, ∗∗P < .01. WT, wild type. Gastroenterology 2018 155, 501-513DOI: (10.1053/j.gastro.2018.04.017) Copyright © 2018 AGA Institute Terms and Conditions

Figure 5 Butyrate decreases obesity in a HDAC3-dependent manner. (A) HDAC activity in IECs from the small intestine of HDAC3FF and HDAC3ΔIEC mice. (B) Nuclear magnetic resonance identification of SCFAs in intestinal contents. (C) HDAC activity and (D) PCK1 mRNA expression in IECs from small intestine explants after vehicle or butyrate treatment. (E, F) Changes in body weight following daily oral gavage with butyrate in (E) wild type or (F) HDAC3ΔIEC mice fed HFD for 20 weeks before and during butyrate exposure. Data represent 3–6 mice per group. Results are shown as mean ± standard error of the mean. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. Gastroenterology 2018 155, 501-513DOI: (10.1053/j.gastro.2018.04.017) Copyright © 2018 AGA Institute Terms and Conditions

Figure 6 HDAC3 in the intestine actively modulates lipid homeostasis. (A) Dietary fat uptake measured using nonabsorbable sucrose polybehenate. (B) Oil Red O stained jejunal tissue from mice fed HFD. Arrowheads indicate Oil Red O stained lipid. (C) Triglyceride levels in IECs of HFD-fed mice. (D) Experimental plan in which HDAC3FF and HDAC3ΔIEC-IND mice were fed diet containing 400mg/kg tamoxifen over 30 days. (E) HDAC3 expression in IECs after tamoxifen. (F) Oil Red O stained jejunal tissue after tamoxifen-induced deletion of HDAC3 in adult mice. Yellow lines represent junction between epithelium and lamina propria. Scale bar = 20 μm. Data represent 4–6 mice per group. Results are shown as mean ± standard error of the mean. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. Gastroenterology 2018 155, 501-513DOI: (10.1053/j.gastro.2018.04.017) Copyright © 2018 AGA Institute Terms and Conditions

Figure 7 Induced disruption of IEC-HDAC3 provides therapeutic effect in obese mice. (A) Experimental plan in which mice were fed HFD for 20 weeks. Tamoxifen was then administered once daily by oral gavage while continuing HFD. (B) Changes in body weight after oral administration of tamoxifen to obese HDAC3FF and HDAC3ΔIEC-IND mice. (C) Experimental plan in which mice were fed HFD for 20 weeks and tamoxifen was administered intraperitoneally to obese HDAC3FF and HDAC3ΔIEC-IND mice over 10 days. (D, E) glucose tolerance in HFD-fed mice (D) before (PRE) and (E) after (POST) tamoxifen. (F) Intestinal epithelial HDAC3 differentially regulates the balance between intestinal and systemic metabolic homeostasis in response to signals from the diet and microbiota. Data represent 4–7 mice per group. Results are shown as mean ± standard error of the mean. ∗P < .05, ∗∗P < .01. d, day; EOD, every other day; min, minute; post, after; pre, before; SID, once a day; Tam, tamoxifen. Gastroenterology 2018 155, 501-513DOI: (10.1053/j.gastro.2018.04.017) Copyright © 2018 AGA Institute Terms and Conditions