Gut Microbiota Remodeling and Intestinal Adaptation to Lipid Malabsorption After Enteroendocrine Cell Loss in Adult Mice.

BACKGROUND & AIMS: Enteroendocrine cells (EECs) and their hormones are essential regulators of whole-body energy homeostasis. EECs sense luminal nutrients and microbial metabolites and subsequently secrete various hormones acting locally or at a distance. Impaired development of EECs during embryogenesis is life-threatening in newborn mice and humans due to compromised nutrient absorption. However, the physiological importance of the EEC system in adult mice has yet to be directedly studied. Herein, we aimed to determine the long-term consequences of a total loss of EECs in healthy adults on energy metabolism, intestinal transcriptome, and microbiota. METHODS: We depleted intestinal EECs by tamoxifen treatment of adult Neurog3fl/fl; Villin-CreERT2 male mice. We studied intestinal cell differentiation, food efficiency, lipid absorption, microbiota composition, fecal metabolites, and transcriptomic responses in the proximal and distal small intestines of mice lacking EECs. We also determined the high-fat diet-induced transcriptomic changes in sorted Neurog3eYFP/+ EECs. RESULTS: Induction of EEC deficiency in adults is not life-threatening unless fed with a high-fat diet. Under a standard chow diet, mice lose 10% of weight due to impaired food efficiency. Blood concentrations of cholesterol, triglycerides, and free fatty acids are reduced, and lipid absorption is impaired and delayed in the distal small intestine. Genes controlling lipogenesis, carbohydrate metabolism, and neoglucogenesis are upregulated. Microbiota composition is rapidly altered after EECs depletion and is characterized by decreased α-diversity. Bacteroides and Lactobacillus were progressively enriched, whereas Lachnospiraceae declined without impacting fecal short-chain fatty acid concentrations. CONCLUSIONS: EECs are dispensable for survival in adult male mice under a standard chow diet. The absence of EECs impairs intestinal lipid absorption, leading to transcriptomic and metabolic adaptations and remodeling of the gut microbiota.

Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network.

OBJECTIVE: Mice lacking the bHLH transcription factor (TF) Neurog3 do not form pancreatic islet cells, including insulin-secreting beta cells, the absence of which leads to diabetes. In humans, homozygous mutations of NEUROG3 manifest with neonatal or childhood diabetes. Despite this critical role in islet cell development, the precise function of and downstream genetic programs regulated directly by NEUROG3 remain elusive. Therefore, we mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (hiPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets. METHODS: We generated a novel hiPSC line (NEUROG3-HA-P2A-Venus) where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) that were differentiated from this hiPSC line. We integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs as well as their NEUROG3-dependence. In addition, we investigated whether NEUROG3 binds type 2 diabetes mellitus (T2DM)-associated variants at the PEP stage. RESULTS: CUT&RUN revealed a total of 863 NEUROG3 binding sites assigned to 1263 unique genes. NEUROG3 occupancy was found at promoters as well as at distant cis-regulatory elements that frequently overlapped within PEP active enhancers. De novo motif analyses defined a NEUROG3 consensus binding motif and suggested potential co-regulation of NEUROG3 target genes by FOXA or RFX transcription factors. We found that 22% of the genes downregulated in NEUROG3-/- PEPs, and 10% of genes enriched in NEUROG3-Venus positive endocrine cells were bound by NEUROG3 and thus likely to be directly regulated. NEUROG3 binds to 138 transcription factor genes, some with important roles in islet cell development or function, such as NEUROD1, PAX4, NKX2-2, SOX4, MLXIPL, LMX1B, RFX3, and NEUROG3 itself, and many others with unknown islet function. Unexpectedly, we uncovered that NEUROG3 targets genes critical for insulin secretion in beta cells (e.g., GCK, ABCC8/KCNJ11, CACNA1A, CHGA, SCG2, SLC30A8, and PCSK1). Thus, analysis of NEUROG3 occupancy suggests that the transient expression of NEUROG3 not only promotes islet destiny in uncommitted pancreatic progenitors, but could also initiate endocrine programs essential for beta cell function. Lastly, we identified eight T2DM risk SNPs within NEUROG3-bound regions. CONCLUSION: Mapping NEUROG3 genome occupancy in PEPs uncovered unexpectedly broad, direct control of the endocrine genes, raising novel hypotheses on how this master regulator controls islet and beta cell differentiation.

Rfx6 promotes the differentiation of peptide-secreting enteroendocrine cells while repressing genetic programs controlling serotonin production.

OBJECTIVE: Enteroendocrine cells (EECs) of the gastro-intestinal tract sense gut luminal factors and release peptide hormones or serotonin (5-HT) to coordinate energy uptake and storage. Our goal is to decipher the gene regulatory networks controlling EECs specification from enteroendocrine progenitors. In this context, we studied the role of the transcription factor Rfx6 which had been identified as the cause of Mitchell-Riley syndrome, characterized by neonatal diabetes and congenital malabsorptive diarrhea. We previously reported that Rfx6 was essential for pancreatic beta cell development and function; however, the role of Rfx6 in EECs differentiation remained to be elucidated. METHODS: We examined the molecular, cellular, and metabolic consequences of constitutive and conditional deletion of Rfx6 in the embryonic and adult mouse intestine. We performed single cell and bulk RNA-Seq to characterize EECs diversity and identify Rfx6-regulated genes. RESULTS: Rfx6 is expressed in the gut endoderm; later, it is turned on in, and restricted to, enteroendocrine progenitors and persists in hormone-positive EECs. In the embryonic intestine, the constitutive lack of Rfx6 leads to gastric heterotopia, suggesting a role in the maintenance of intestinal identity. In the absence of intestinal Rfx6, EECs differentiation is severely impaired both in the embryo and adult. However, the number of serotonin-producing enterochromaffin cells and mucosal 5-HT content are increased. Concomitantly, Neurog3-positive enteroendocrine progenitors accumulate. Combined analysis of single-cell and bulk RNA-Seq data revealed that enteroendocrine progenitors differentiate in two main cell trajectories, the enterochromaffin (EC) cells and the Peptidergic Enteroendocrine (PE) cells, the differentiation programs of which are differentially regulated by Rfx6. Rfx6 operates upstream of Arx, Pax6 and Isl1 to trigger the differentiation of peptidergic EECs such as GIP-, GLP-1-, or CCK-secreting cells. On the contrary, Rfx6 represses Lmx1a and Tph1, two genes essential for serotonin biosynthesis. Finally, we identified transcriptional changes uncovering adaptive responses to the prolonged lack of enteroendocrine hormones and leading to malabsorption and lower food efficiency ratio in Rfx6-deficient mouse intestine. CONCLUSION: These studies identify Rfx6 as an essential transcriptional regulator of EECs specification and shed light on the molecular mechanisms of intestinal failures in human RFX6-deficiencies such as Mitchell-Riley syndrome.

Expression and functional studies of the GDNF family receptor alpha 3 in the pancreas.

The generation of therapeutic ß-cells from human pluripotent stem cells relies on the identification of growth factors that faithfully mimic pancreatic ß-cell development in vitro. In this context, the aim of the study was to determine the expression and function of the glial cell line derived neurotrophic factor receptor alpha 3 (GFRα3) and its ligand artemin (Artn) in islet cell development and function. GFRα3 and Artn expression were characterized by in situ hybridization, immunochemistry, and qRT-PCR. We used GFRα3-deficient mice to study GFRα3 function and generated transgenic mice overexpressing Artn in the embryonic pancreas to study Artn function. We found that GFRα3 is expressed at the surface of a subset of Ngn3-positive endocrine progenitors as well as of embryonic α- and ß-cells, while Artn is found in the pancreatic mesenchyme. Adult ß-cells lack GFRα3 but α-cells express the receptor. GFRα3 was also found in parasympathetic and sympathetic intra-islet neurons as well as in glial cells in the embryonic and adult pancreas. The loss of GFRα3 or overexpression of Artn has no impact on Ngn3 and islet cell formation and maintenance in the embryo. Islet organization and innervation as well as glucose homeostasis is normal in GFRα3-deficient mice suggesting functional redundancy.

Rfx6 maintains the functional identity of adult pancreatic ß cells.

Increasing evidence suggests that loss of ß cell characteristics may cause insulin secretory deficiency in diabetes, but the underlying mechanisms remain unclear. Here, we show that Rfx6, whose mutation leads to neonatal diabetes in humans, is essential to maintain key features of functionally mature ß cells in mice. Rfx6 loss in adult ß cells leads to glucose intolerance, impaired ß cell glucose sensing, and defective insulin secretion. This is associated with reduced expression of core components of the insulin secretion pathway, including glucokinase, the Abcc8/SUR1 subunit of KATP channels and voltage-gated Ca(2+) channels, which are direct targets of Rfx6. Moreover, Rfx6 contributes to the silencing of the vast majority of "disallowed" genes, a group usually specifically repressed in adult ß cells, and thus to the maintenance of ß cell maturity. These findings raise the possibility that changes in Rfx6 expression or activity may contribute to ß cell failure in humans.

Pak3 promotes cell cycle exit and differentiation of ß-cells in the embryonic pancreas and is necessary to maintain glucose homeostasis in adult mice.

The transcription factor neurogenin3 (Ngn3) triggers islet cell differentiation in the developing pancreas. However, little is known about the molecular mechanisms coupling cell cycle exit and differentiation in Ngn3(+) islet progenitors. We identified a novel effector of Ngn3 endocrinogenic function, the p21 protein-activated kinase Pak3, known to control neuronal differentiation and implicated in X-linked intellectual disability in humans. We show that Pak3 expression is initiated in Ngn3(+) endocrine progenitor cells and next maintained in maturing hormone-expressing cells during pancreas development as well as in adult islet cells. In Pak3-deficient embryos, the proliferation of Ngn3(+) progenitors and ß-cells is transiently increased concomitantly with an upregulation of Ccnd1. ß-Cell differentiation is impaired at E15.5 but resumes at later stages. Pak3-deficient mice do not develop overt diabetes but are glucose intolerant under high-fat diet (HFD). In the intestine, Pak3 is expressed in enteroendocrine cells but is not necessary for their differentiation. Our results indicate that Pak3 is a novel regulator of ß-cell differentiation and function. Pak3 acts downstream of Ngn3 to promote cell cycle exit and differentiation in the embryo by a mechanism that might involve repression of Ccnd1. In the adult, Pak3 is required for the proper control of glucose homeostasis under challenging HFD.

The homeodomain-containing transcription factors Arx and Pax4 control enteroendocrine subtype specification in mice.

Intestinal hormones are key regulators of digestion and energy homeostasis secreted by rare enteroendocrine cells. These cells produce over ten different hormones including GLP-1 and GIP peptides known to promote insulin secretion. To date, the molecular mechanisms controlling the specification of the various enteroendocrine subtypes from multipotent Neurog3(+) endocrine progenitor cells, as well as their number, remain largely unknown. In contrast, in the embryonic pancreas, the opposite activities of Arx and Pax4 homeodomain transcription factors promote islet progenitor cells towards the different endocrine cell fates. In this study, we thus investigated the role of Arx and Pax4 in enteroendocrine subtype specification. The small intestine and colon of Arx- and Pax4-deficient mice were analyzed using histological, molecular, and lineage tracing approaches. We show that Arx is expressed in endocrine progenitors (Neurog3(+)) and in early differentiating (ChromograninA(-)) GLP-1-, GIP-, CCK-, Sct- Gastrin- and Ghrelin-producing cells. We noted a dramatic reduction or a complete loss of all these enteroendocrine cell types in Arx mutants. Serotonin- and Somatostatin-secreting cells do not express Arx and, accordingly, the differentiation of Serotonin cells was not affected in Arx mutants. However, the number of Somatostatin-expressing D-cells is increased as Arx-deficient progenitor cells are redirected to the D-cell lineage. In Pax4-deficient mice, the differentiation of Serotonin and Somatostatin cells is impaired, as well as of GIP and Gastrin cells. In contrast, the number of GLP-1 producing L-cells is increased concomitantly with an upregulation of Arx. Thus, while Arx and Pax4 are necessary for the development of L- and D-cells respectively, they conversely restrict D- and L-cells fates suggesting antagonistic functions in D/L cell allocation. In conclusion, these finding demonstrate that, downstream of Neurog3, the specification of a subset of enteroendocrine subtypes relies on both Arx and Pax4, while others depend only on Arx or Pax4.