Quantifying Forms and Functions of Enterohepatic Bile Acid Pools in Mice.

BACKGROUNDS & AIMS: Bile acids (BAs) are core gastrointestinal metabolites with dual functions in lipid absorption and cell signaling. BAs circulate between the liver and distal small intestine (ie, ileum), yet the dynamics through which complex BA pools are absorbed in the ileum and interact with host intestinal cells in vivo remain poorly understood. Because ileal absorption is rate-limiting in determining which BAs in the intestinal lumen gain access to host intestinal cells and receptors, and at what concentrations, we hypothesized that defining the rates and routes of ileal BA absorption in vivo would yield novel insights into the physiological forms and functions of mouse enterohepatic BA pools. METHODS: Using ex vivo mass spectrometry, we quantified 88 BA species and metabolites in the intestinal lumen and superior mesenteric vein of individual wild-type mice, and cage-mates lacking the ileal BA transporter, Asbt/Slc10a2. RESULTS: Using these data, we calculated that the pool of BAs circulating through ileal tissue (ie, the ileal BA pool) in fasting C57BL/6J female mice is ∼0.3 mmol/g. Asbt-mediated transport accounted for ∼80% of this pool and amplified size. Passive permeability explained the remaining ∼20% and generated diversity. Compared with wild-type mice, the ileal BA pool in Asbt-deficient mice was ∼5-fold smaller, enriched in secondary BA species and metabolites normally found in the colon, and elicited unique transcriptional responses on addition to ex vivo-cultured ileal explants. CONCLUSIONS: This study defines quantitative traits of the mouse enterohepatic BA pool and reveals how aberrant BA metabolism can impinge directly on host intestinal physiology.

An immune-adrenergic pathway induces lethal levels of platelet-activating factor in mice.

Acute immune responses with excess production of cytokines, lipid/chemical mediators, or coagulation factors, often result in lethal damage. In addition, the innate immune system utilizes multiple types of receptors that recognize neurotransmitters as well as pathogen-associated molecular patterns, making immune responses complex and clinically unpredictable. We here report an innate immune and adrenergic link inducing lethal levels of platelet-activating factor. Injecting mice with toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS), cell wall N-glycans of Candida albicans, and the α2-adrenergic receptor (α2-AR) agonist medetomidine induces lethal damage. Knocking out the C-type lectin Dectin-2 prevents the lethal damage. In spleen, large amounts of platelet-activating factor (PAF) are detected, and knocking out lysophospholipid acyltransferase 9 (LPLAT9/LPCAT2), which encodes an enzyme that converts inactive lyso-PAF to active PAF, protects mice from the lethal damage. These results reveal a linkage/crosstalk between the nervous and the immune system, possibly inducing lethal levels of PAF.

Quantifying forms and functions of intestinal bile acid pools in mice.

Bile acids (BAs) are gastrointestinal metabolites that serve dual functions in lipid absorption and cell signaling. BAs circulate actively between the liver and distal small intestine (i.e., ileum), yet the dynamics through which complex BA pools are absorbed in the ileum and interact with intestinal cells in vivo remain ill-defined. Through multi-site sampling of nearly 100 BA species in individual wild type mice, as well as mice lacking the ileal BA transporter, Asbt/Slc10a2, we calculate the ileal BA pool in fasting C57BL/6J mice to be ~0.3 µmoles/g. Asbt-mediated transport accounts for ~80% of this pool and amplifies size, whereas passive absorption explains the remaining ~20%, and generates diversity. Accordingly, ileal BA pools in mice lacking Asbt are ~5-fold smaller than in wild type controls, enriched in secondary BA species normally found in the colon, and elicit unique transcriptional responses in cultured ileal explants. This work quantitatively defines ileal BA pools in mice and reveals how BA dysmetabolism can impinge on intestinal physiology.

Vγ5Vδ1 TCR signaling is required to different extents for embryonic versus postnatal development of DETCs.

γδ T cells expressing Vγ5Vδ1 TCR originally develop in the embryonic thymus and migrate to the epidermis, forming dendritic epidermal T cells (DETCs) throughout life. It is thought that a TCR signal is essential for their development; e.g., lack of TCR signal-transducer ZAP70 significantly decreases DETC numbers. On the other hand, lack of ZAP70 does not affect Vγ5Vδ1+ T cells in the embryonic thymus; thus, the involvement of TCR signaling remains elusive. Here, we used SKG mice with attenuated TCR signaling rather than gene-knockout mice. In SKG mice, Vγ5+ T cells showed a marked decrease [10% of wild-type (WT)] in adult epidermis; however, there was just a moderate decrease (50% of WT) in the embryonic thymus. In early postnatal epidermis in SKG mice, substantial numbers of Vγ5+ T cells were observed (50% of WT). Their activation markers including CD122, a component of the IL-15 receptor indispensable for DETC proliferation, were comparable to those of WT. However, the Vγ5+ T cells in SKG mice did not proliferate and form DETCs thereafter. Furthermore, in SKG/+ mice, the number of thymic Vγ5Vδ1+ T cells increased, compared to SKG mice; however, the number of DETCs remained significantly lower than in WT, similar to SKG mice. Our results suggest that signaling via Vγ5Vδ1 TCR is indispensable for DETC development, with distinct contributions to embryonic development and postnatal proliferation.

Recombinant Mouse Osteocalcin Secreted by Lactococcus lactis Promotes Glucagon-Like Peptide-1 Induction in STC-1 Cells.

An osteoblastic protein, osteocalcin (OC), exists in vivo in two forms: carboxylated OC, and uncarboxylated or low-carboxylated OC (ucOC). ucOC acts as a hormone to regulate carbon and energy metabolism. Recent studies demonstrated that ucOC exerts insulinotropic effects, mainly through the glucagon-like peptide 1 (GLP-1) pathway. GLP-1 is an insulinotropic hormone secreted by enteroendocrine L cells in the small intestine. Thus, efficient delivery of ucOC to the small intestine may be a new therapeutic option for metabolic diseases such as diabetes and obesity. Here, we genetically engineered a lactic acid bacterium, Lactococcus lactis, to produce recombinant mouse ucOC. Western blotting showed that the engineered strain (designated NZ-OC) produces and secretes the designed peptide (rOC) in the presence of nisin, an inducer of the recombinant gene. Highly-purified rOC was obtained from the culture supernatants of NZ-OC using immobilized metal affinity chromatography. An in vitro assay showed that purified rOC promotes GLP-1 secretion in a mouse intestinal neuroendocrine cell line, STC-1, in a dose-dependent manner. These results clearly demonstrate that NZ-OC secretes rOC, and that rOC can promote GLP-1 secretion by STC-1 cells. Genetically modified lactic acid bacteria (gmLAB) have been proposed over the last two decades as an effective and low-cost mucosal delivery vehicle for biomedical proteins. NZ-OC may be an attractive tool for the delivery of rOC to trigger GLP-1 secretion in the small intestine to treat diabetes and obesity.