Department of Genetics, Human Genetics Institute of New Jersey, Rutgers University, Piscataway, New Jersey; Rutgers Cancer Institute of New Jersey, New Brunswick, New Jersey.
Department of Genetics, Human Genetics Institute of New Jersey, Rutgers University, Piscataway, New Jersey.
Gastroenterology. 2020 Mar;158(4):985-999.e9. doi: 10.1053/j.gastro.2019.11.031. Epub 2019 Nov 22.
BACKGROUND & AIMS: Functions of intestinal stem cells (ISCs) are regulated by diet and metabolic pathways. Hepatocyte nuclear factor 4 (HNF4) family are transcription factors that bind fatty acids. We investigated how HNF4 transcription factors regulate metabolism and their functions in ISCs in mice.
We performed studies with Villin-Cre;Lgr5-EGFP-IRES-Cre;Hnf4α;Hnf4γ mice, hereafter referred to Hnf4αγ. Mice were given tamoxifen to induce Cre recombinase. Mice transgenic with only Cre alleles (Villin-Cre, Lgr5-EGFP-IRES-Cre, Hnf4α, and Hnf4γ) or mice given vehicle were used as controls. Crypt and villus cells were isolated, incubated with fluorescently labeled fatty acids or glucose analog, and analyzed by confocal microscopy. Fatty acid oxidation activity and tricarboxylic acid (TCA) cycle metabolites were measured in cells collected from the proximal half of the small intestine of Hnf4αγ and control mice. We performed chromatin immunoprecipitation and gene expression profiling analyses to identify genes regulated by HNF4 factors. We established organoids from duodenal crypts, incubated them with labeled palmitate or acetate, and measured production of TCA cycle metabolites or fatty acids. Acetate, a precursor of acetyl coenzyme A (CoA) (a product of fatty acid β-oxidation [FAO]), or dichloroacetate, a compound that promotes pyruvate oxidation and generation of mitochondrial acetyl-CoA, were used for metabolic intervention.
Crypt cells rapidly absorbed labeled fatty acids, and messenger RNA levels of Lgr5 stem cell markers (Lgr5, Olfm4, Smoc2, Msi1, and Ascl2) were down-regulated in organoids incubated with etomoxir, an inhibitor of FAO, indicating that FAO was required for renewal of ISCs. HNF4A and HNF4G were expressed in ISCs and throughout the intestinal epithelium. Single knockout of either HNF4A or HNF4G did not affect maintenance of ISCs, but double-knockout of HNF4A and HNF4G resulted in ISC loss; stem cells failed to renew. FAO supports ISC renewal, and HNF4 transcription factors directly activate FAO genes, including Acsl5 and Acsf2 (encode regulators of acyl-CoA synthesis), Slc27a2 (encodes a fatty acid transporter), Fabp2 (encodes fatty acid binding protein), and Hadh (encodes hydroxyacyl-CoA dehydrogenase). In the intestinal epithelium of Hnf4αγ mice, expression levels of FAO genes, FAO activity, and metabolites of TCA cycle were all significantly decreased, but fatty acid synthesis transcripts were increased, compared with control mice. The contribution of labeled palmitate or acetate to the TCA cycle was reduced in organoids derived from Hnf4αγ mice, compared with control mice. Incubation of organoids derived from double-knockout mice with acetate or dichloroacetate restored stem cells.
In mice, the transcription factors HNF4A and HNF4G regulate the expression of genes required for FAO and are required for renewal of ISCs.
肠干细胞（ISCs）的功能受饮食和代谢途径的调节。肝细胞核因子 4（HNF4）家族是与脂肪酸结合的转录因子。我们研究了 HNF4 转录因子如何调节代谢及其在小鼠 ISCs 中的功能。
我们使用 Villin-Cre；Lgr5-EGFP-IRES-Cre；Hnf4α；Hnf4γ 小鼠（以下简称 Hnf4αγ）进行了研究。给予他莫昔芬以诱导 Cre 重组酶。使用仅携带 Cre 等位基因（Villin-Cre、Lgr5-EGFP-IRES-Cre、Hnf4α 和 Hnf4γ）的转基因小鼠或给予载体的小鼠作为对照。分离隐窝和绒毛细胞，用荧光标记的脂肪酸或葡萄糖类似物孵育，并通过共聚焦显微镜进行分析。测量 Hnf4αγ 和对照小鼠近端小肠细胞中的脂肪酸氧化活性和三羧酸（TCA）循环代谢物。我们进行了染色质免疫沉淀和基因表达谱分析，以鉴定受 HNF4 因子调节的基因。我们从十二指肠隐窝建立了类器官，用标记的棕榈酸或醋酸盐孵育，并测量 TCA 循环代谢物或脂肪酸的产生。醋酸盐是乙酰辅酶 A（CoA）的前体（脂肪酸 β-氧化[FAO]的产物），或二氯乙酸盐，一种促进丙酮酸氧化和生成线粒体乙酰 CoA 的化合物，用于代谢干预。
隐窝细胞迅速吸收标记的脂肪酸，用 etomoxir（FAO 的抑制剂）孵育的类器官中 Lgr5 干细胞标志物（Lgr5、Olfm4、Smoc2、Msi1 和 Ascl2）的信使 RNA 水平下调，表明 FAO 是 ISC 更新所必需的。HNF4A 和 HNF4G 在 ISCs 和整个肠上皮中表达。单独敲除 HNF4A 或 HNF4G 不会影响 ISC 的维持，但双敲除 HNF4A 和 HNF4G 会导致 ISC 丢失；干细胞无法更新。FAO 支持 ISC 更新，HNF4 转录因子直接激活 FAO 基因，包括 Acsl5 和 Acsf2（编码酰基辅酶 A 合成调节剂）、Slc27a2（编码脂肪酸转运蛋白）、Fabp2（编码脂肪酸结合蛋白）和 Hadh（编码羟基酰基辅酶 A 脱氢酶）。与对照小鼠相比，Hnf4αγ 小鼠的肠上皮中 FAO 基因的表达水平、FAO 活性和 TCA 循环代谢物均显著降低，但脂肪酸合成转录本增加。与对照小鼠相比，源自 Hnf4αγ 小鼠的类器官中标记的棕榈酸或醋酸盐对 TCA 循环的贡献减少。用醋酸盐或二氯乙酸盐孵育源自双敲除小鼠的类器官可恢复干细胞。
在小鼠中，转录因子 HNF4A 和 HNF4G 调节 FAO 所需基因的表达，是 ISC 更新所必需的。
