Identification and Distribution of Sterols, Bile Acids, and Acylcarnitines by LC–MS/MS in Humans, Mice, and Pigs—A Qualitative Analysis

Sterols, bile acids, and acylcarnitines are key players in human metabolism. Precise annotations of these metabolites with mass spectrometry analytics are challenging because of the presence of several isomers and stereoisomers, variability in ionization, and their relatively low concentrations in biological samples. Herein, we present a sensitive and simple qualitative LC–MS/MS (liquid chromatography with tandem mass spectrometry) method by utilizing a set of pure chemical standards to facilitate the identification and distribution of sterols, bile acids, and acylcarnitines in biological samples including human stool and plasma; mouse ileum, cecum, jejunum content, duodenum content, and liver; and pig bile, proximal colon, cecum, heart, stool, and liver. With this method, we detected 24 sterol, 32 bile acid, and 27 acylcarnitine standards in one analysis that were separated within 13 min by reversed-phase chromatography. Further, we observed different sterol, bile acid, and acylcarnitine profiles for the different biological samples across the different species. The simultaneous detection and annotation of sterols, bile acids, and acylcarnitines from reference standards and biological samples with high precision represents a valuable tool for screening these metabolites in routine scientific research.

A temporal Ca2+-desensitization of myosin light chain kinase in phasic smooth muscles induced by CaMKKß/PP2A pathways.

Cell signaling pathways regulating myosin regulatory light chain (LC20) phosphorylation contribute to determining contractile responses in smooth muscles. Following excitation and contraction, phasic smooth muscles, such as digestive tract and urinary bladder, undergo a relaxation due to a decline of cellular [Ca2+] and a decreased Ca2+ sensitivity of LC20 phosphorylation, named Ca2+ desensitization. Here, we determined mechanisms underlying the temporal Ca2+ desensitization of LC20 phosphorylation in phasic smooth muscles using permeabilized strips of mouse ileum and urinary bladder. Upon the stimulation with pCa6.0 at 20°C, the contraction and the LC20 phosphorylation peaked within 30 sec and then declined to about 50% of the peak force at 2 min after stimulation. During the relaxation phase after the contraction, the LC20 kinase (MLCK) was inactivated, but no fluctuation in the LC20 phosphatase activity occurred, suggesting that the MLCK inactivation is a cause of the Ca2+-induced Ca2+-desensitization of LC20 phosphorylation. The MLCK inactivation was associated with phosphorylation at the calmodulin binding domain of the kinase. Treatment with antagonists for CaMKKß (STO-609 and TIM-063) attenuated both the phasic response of the contraction and MLCK phosphorylation, whereas neither CaMKII, AMPK nor PAK induced the MLCK inactivation in phasic smooth muscles. Conversely, PP2A inhibition amplified the phasic response. Signaling pathways through CaMKKß and PP2A may contribute to regulating the Ca2+ sensitivity of MLCK and the contractile response of phasic smooth muscles.

NFAT5 Is Involved in GRP-Enhanced Secretion of GLP-1 by Sodium

Gastrin, secreted by G-cells, and glucagon-like peptide-1 (GLP-1), secreted by L-cells, may participate in the regulation of sodium balance. We studied the effect of sodium in mice in vivo and mouse ileum and human L-cells, on GLP-1 secretion, and the role of NFAT5 and gastrin-releasing peptide receptor (GRPR) in this process. A high-sodium diet increases serum GLP-1 levels in mice. Increasing sodium concentration stimulates GLP-1 secretion from mouse ileum and L-cells. GRP enhances the high sodium-induced increase in GLP-1 secretion. High sodium increases cellular GLP-1 expression, while low and high sodium concentrations increase NFAT5 and GRPR expression. Silencing NFAT5 in L-cells abrogates the stimulatory effect of GRP on the high sodium-induced GLP-1 secretion and protein expression, and the sodium-induced increase in GRPR expression. GLP-1 and gastrin decrease the expression of Na+-K+/ATPase and increase the phosphorylation of sodium/hydrogen exchanger type 3 (NHE3) in human renal proximal tubule cells (hRPTCs). This study gives a new perspective on the mechanisms of GLP-1 secretion, especially that engendered by ingested sodium, and the ability of GLP-1, with gastrin, to decrease Na+-K+/ATPase expression and NHE3 function in hRPTCs. These results may contribute to the better utilization of current and future GLP-1-based drugs in the treatment of hypertension.

The Intestinal Efflux Transporter Inhibition Activity of Xanthones from Mangosteen Pericarp: An In Silico, In Vitro and Ex Vivo Approach

The capacity of α-mangostin (α-MG) and β-mangostin (β-MG) from mangosteen pericarp on P-glycoprotein (Pgp) in silico, in vitro, and ex vivo was investigated in this study. Screening with the ADMET Predictor™ program predicted the two compounds to be both a Pgp inhibitor and Pgp substrate. The compounds tended to interact with Pgp and inhibit Pgp ATPase activity. Additionally, bidirectional transport on Caco-2 cell monolayers demonstrated a significantly lower efflux ratio than that of the control (α-(44.68) and β-(46.08) MG versus the control (66.26); p < 0.05) indicating an inhibitory effect on Pgp activity. Test compounds additionally revealed a downregulation of MDR1 mRNA expression. Moreover, an ex vivo absorptive transport in everted mouse ileum confirmed the previous results that α-MG had a Pgp affinity inhibitor, leading to an increase in absorption of the Pgp substrate in the serosal side. In conclusion, α- and β-MG have the capability to inhibit Pgp and they also alter Pgp expression, which makes them possible candidates for reducing multidrug resistance. Additionally, they influence the bioavailability and transport of Pgp substrate drugs.

Bile acid composition regulates the manganese transporter Slc30a10 in intestine

Bile acids (BAs) comprise heterogenous amphipathic cholesterol-derived molecules that carry out physicochemical and signaling functions. A major site of BA action is the terminal ileum, where enterocytes actively reuptake BAs and express high levels of BA-sensitive nuclear receptors. BA pool size and composition are affected by changes in metabolic health, and vice versa. One of several factors that differentiate BAs is the presence of a hydroxyl group on C12 of the steroid ring. 12α-Hydroxylated BAs (12HBAs) are altered in multiple disease settings, but the consequences of 12HBA abundance are incompletely understood. We employed mouse primary ileum organoids to investigate the transcriptional effects of varying 12HBA abundance in BA pools. We identified Slc30a10 as one of the top genes differentially induced by BA pools with varying 12HBA abundance. SLC30A10 is a manganese efflux transporter critical for whole-body manganese excretion. We found that BA pools, especially those low in 12HBAs, induce cellular manganese efflux and that Slc30a10 induction by BA pools is driven primarily by lithocholic acid signaling via the vitamin D receptor. Administration of lithocholic acid or a vitamin D receptor agonist resulted in increased Slc30a10 expression in mouse ileum epithelia. These data demonstrate a previously unknown role for BAs in intestinal control of manganese homeostasis.

Acetylcholine exerts inhibitory and excitatory actions on mouse ileal pacemaker activity: role of muscarinic versus nicotinic receptors

The study discovered an acute action of acetylcholine on pacemaker potentials that is mediated by muscarinic receptors on the mouse ileum. Bethanechol, but not nicotine, mimicked the inhibitory actions of acetylcholine on pacemaker potentials. Atropine, but not hexamethonium, reversed the inhibitory actions of acetylcholine. When introduced after acetylcholine, atropine exhibited excitatory actions that increased the pacemaker frequency. Acetylcholine and bethanechol distorted the propagation activity and pattern, and this was also reversed by atropine. These actions of acetylcholine on pacemaker potentials may contribute to pathophysiology in bowel diseases.

Involvement of Enteric Glia in Small Intestine Neuromuscular Dysfunction of Toll-Like Receptor 4-Deficient Mice

Enteric glial cells (EGCs) influence nitric oxide (NO)− and adenosine diphosphate (ADP)− mediated signaling in the enteric nervous system (ENS). Since Toll-like receptor 4 (TLR4) participates to EGC homoeostasis, this study aimed to evaluate the possible involvement of EGCs in the alterations of the inhibitory neurotransmission in TLR4−/− mice. Ileal segments from male TLR4−/− and wild-type (WT) C57BL/6J mice were incubated with the gliotoxin fluoroacetate (FA). Alterations in ENS morphology and neurochemical coding were investigated by immunohistochemistry whereas neuromuscular responses were determined by recording non-adrenergic non-cholinergic (NANC) relaxations in isometrically suspended isolated ileal preparations. TLR4−/− ileal segments showed increased iNOS immunoreactivity associated with enhanced NANC relaxation, mediated by iNOS-derived NO and sensitive to P2Y1 inhibition. Treatment with FA diminished iNOS immunoreactivity and partially abolished NO− and ADP− mediated relaxation in the TLR4−/− mouse ileum, with no changes of P2Y1 and connexin-43 immunofluorescence distribution in the ENS. After FA treatment, S100β and GFAP immunoreactivity in TLR4−/− myenteric plexus was reduced to levels comparable to those observed in WT. Our findings show the involvement of EGCs in the alterations of ENS architecture and in the increased purinergic and nitrergic-mediated relaxation, determining gut dysmotility in TLR4−/− mice.