Global Mapping of Cell Type–Specific Open Chromatin by FAIRE-seq Reveals the Regulatory Role of the NFI Family in Adipocyte Differentiation

Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type–specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation) and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI) transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA–mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our study demonstrates the utility of FAIRE-seq in providing a global view of cell type–specific regulatory elements in the genome and in identifying transcriptional regulators of adipocyte differentiation

Pharmacological Inhibition of Monoacylglycerol O-Acyltransferase 2 Improves Hyperlipidemia, Obesity, and Diabetes by Change in Intestinal Fat Utilization

<div><p>Monoacylglycerol O-acyltransferase 2 (MGAT2) catalyzes the synthesis of diacylglycerol (DG), a triacylglycerol precursor and potential peripheral target for novel anti-obesity therapeutics. High-throughput screening identified lead compounds with MGAT2 inhibitory activity. Through structural modification, a potent, selective, and orally bioavailable MGAT2 inhibitor, compound A (compA), was discovered. CompA dose-dependently inhibited postprandial increases in plasma triglyceride (TG) levels. Metabolic flux analysis revealed that compA inhibited triglyceride/diacylglycerol resynthesis in the small intestine and increased free fatty acid and acyl-carnitine with shorter acyl chains than originally labelled fatty acid. CompA decreased high-fat diet (HFD) intake in C57BL/6J mice. MGAT2-null mice showed a similar phenotype as compA-treated mice and compA did not suppress a food intake in MGAT2 KO mice, indicating that the anorectic effects were dependent on MGAT2 inhibition. Chronic administration of compA significantly prevented body weight gain and fat accumulation in mice fed HFD. MGAT2 inhibition by CompA under severe diabetes ameliorated hyperglycemia and fatty liver in HFD-streptozotocin (STZ)-treated mice. Homeostatic model assessments (HOMA-IR) revealed that compA treatment significantly improved insulin sensitivity. The proximal half of the small intestine displayed weight gain following compA treatment. A similar phenomenon has been observed in Roux-en-Y gastric bypass-treated animals and some studies have reported that this intestinal remodeling is essential to the anti-diabetic effects of bariatric surgery. These results clearly demonstrated that MGAT2 inhibition improved dyslipidemia, obesity, and diabetes, suggesting that compA is an effective therapeutic for obesity-related metabolic disorders.</p></div

Effect of compound A (compA) on postprandial triglyceride (TG) excursion.

<p>Fasted C57BL/6J mice were given a liquid meal orally with intraperitoneal injection of Pluronic F-127 to inhibit plasma TG lipolysis. (A) Structure of compA. Plasma samples were collected at 0, 2, and 4 h after oral gavage of a liquid meal. (B) Time course of changes in plasma chylomicron TG (CM/TG) levels and (C) postprandial TG excursion of 3 or 10 mg/kg compA at 6 h after dosing. (D) Time course of changes in plasma TG levels and (E) postprandial TG excursion of 30 mg/kg compA at 16 h after dosing. n = 6 (B, C), and n = 7 (D, E). #: <i>P</i> < 0.025 vs. vehicle group by one-tailed Williams’ test. **: <i>P</i> < 0.01, ***: <i>P</i> < 0.001 vs. vehicle group by Student’s t-test.</p

Intestinal remodeling by compA in high-fat diet-streptozotocin (HFD-STZ) mice.

<p>The small intestine of HFD-STZ mice was collected after 6-week administration of 30 mg/kg compA. Mean (A) tissue weight of the upper and (B) lower halves of the small intestine. Mean levels of gene expression for select enzymes involved in cholesterol metabolism in the (C) upper and (D) lower halves of the small intestine. n = 7 (for the weight of the upper intestine) or n = 8. *: <i>P</i> < 0.05, **: <i>P</i> < 0.01, ***: <i>P</i> < 0.001 vs. the vehicle-treated group by Student’s t-test. &: <i>P</i> < 0.05 vs. the vehicle-treated group by Aspin–Welch test.</p

Acute anorectic effect of compA under high-fat diet (HFD)-feeding conditions.

<p>Fasted mice were orally administrated vehicle or compA and were fed either HFD or normal chow (NC) for 2 h. (A) Amount of HFD intake by C57BL/6J mice. (B) Amount of HFD intake by vehicle or 10 mg/kg compA-treated MGAT2 KO mice and WT littermates. (C) Amount of NC or HFD intake by vehicle or 10 mg/kg compA-treated C57BL/6J mice. n = 5 (A, B) or n = 7 (C). #: <i>P</i> < 0.025 vs. vehicle group by one-tailed Williams’ test. %%%: <i>P</i> < 0.001 vs. vehicle-administrated WT mice by Student’s t-test. *: <i>P</i> < 0.05 vs. HFD-fed vehicle group by Student's t-test. N.S.: not significant.</p

Lipid metabolic flux analysis of compA in intestinal mucosa.

<p>Fasted C57BL/6J mice were orally administered 30 mg/kg compA. Two hours after dosing, they were orally administered a liquid meal containing the ²H-labeled oleoylglycerol. Glycerol-labeled monoacylglycerol (MG) was used in panels 2A and 2C, and fatty acid-labeled MG was used in panel 2B as a tracer. (A) Intestinal ²H-labeled MG, diacylglycerol (DG), and triglyceride (TG) levels. (B) Intestinal ²H-labeled fatty acid and acylcarnitine levels. (C) Intestinal ²H-labeled phospholipids levels. n = 5. **: <i>P</i> < 0.01, ***: <i>P</i> < 0.001 vs. vehicle group by Student's t-test. &: <i>P</i> < 0.05, &&: <i>P</i> < 0.01, &&&: <i>P</i> < 0.001 vs. vehicle group by Aspin–Welch test. (D) Scheme of MGAT2 inhibitor-mediated intestinal fat metabolism. (1) Most of the dietary MG and free fatty acids (FFAs) is used as a source of TG resynthesis in enterocytes. Some of the synthesized DGs are pooled as phospholipids. (2) MGAT2 inhibitor suppresses DG/TG resynthesis. Transiently stored MG is hydrolyzed into glycerol and FFAs. The activated FFA (acylcarnitine) is transported into mitochondria as a substrate of β-oxidation.</p

Anti-obesity effects of compA in C57BL/6J mice fed a high-fat diet (HFD).

<p>Male C57BL/6J mice were fed HFD and orally administered vehicle, 30 mg/kg compA, or 10 mg/kg sibutramine for 5 weeks. (A) Time course of changes in body weight (BW). (B) Food intake. Mean (C) fat mass and (D) lean mass. n = 6. *: <i>P</i> < 0.05, ***: <i>P</i> < 0.001 vs. WT mice by two-tailed Dunnett’s test. The results of Fig 4D were analyzed using two-tailed Steel’s test. (E) GLUTag cells were treated with assay buffer containing free fatty acid (oleic acid), monoacylglycerol (2-monoacylglycerol), diacylglycerol (diolein), and triglyceride (triolein) for 2h. Secreted GLP-1 levels in the assay buffer was measured by GLP-1 ELISA. n = 4. #: P < 0.025 vs. control by one-tailed Williams test.</p