Quantitative Determination of Cholesterol Hydroxylase Specificities by GC-MS/MS in Living Mammalian Cells.

Cholesterol is oxygenated by a variety of cholesterol hydroxylases; oxysterols play diverse important roles in physiological and pathophysiological conditions by regulating several transcription factors and cell-surface receptors. Each oxysterol has distinct and overlapping functions. The expression of cholesterol hydroxylases is highly regulated, but their physiological and pathophysiological roles are not fully understood. Although the activity of cholesterol hydroxylases has been characterized biochemically using radiolabeled cholesterol as the substrate, their specificities remain to be comprehensively determined quantitatively. To better understand their roles, a highly sensitive method to measure the amount of various oxysterols synthesized by cholesterol hydroxylases in living mammalian cells is required. Our method described here, with gas chromatography coupled with tandem mass spectrometry (GC-MS/MS), can quantitatively determine a series of oxysterols endogenously synthesized by forced expression of one of the four major cholesterol hydroxylases-CH25H, CYP7A1, CYP27A1, and CYP46A1-or induction of CH25H expression by a physiological stimulus. This protocol can also simultaneously measure the amount of intermediate sterols, which serve as markers for cellular cholesterol synthesis activity. Key features • Allows measuring the amount of a variety of oxysterols synthesized endogenously by cholesterol hydroxylases using GC-MS/MS. • Comprehensive and quantitative analysis of cholesterol hydroxylase specificities in living mammalian cells. • Simultaneous quantification of intermediate sterols to assess cholesterol synthesis activity.

Establishment of a cell culture platform for human liver organoids and its application for lipid metabolism research.

Human liver organoids (HLOs) are reliable tools to represent physiological human liver biology. However, their use is limited especially in basic sciences. One of the reasons for this would be the insufficient systematic methodology to handle HLOs, including culture system, functional assessment, and gene transduction. Here, we generated and characterized mouse L cells stably and simultaneously overexpressing R-spondin1, hepatocyte growth factor, fibroblast growth factor (FGF) 7, and FGF10 via lentiviral transduction. The conditioned medium of the cells contributed to HLO growth as a replacement of commercially available recombinant proteins, which leads to a significant reduction of their culture cost. Proliferative and maturation phases of the cells were controlled by switching the medium to facilitate the evaluation of hepatocyte function, including insulin responsiveness and intracellular lipid accumulation. Gene expression analysis revealed that HLOs highly expressed genes involved in lipid metabolism. Importantly, HLOs secreted physiologically matured very low-density lipoprotein, which is rarely observed in mice and in established cell lines. Efficient gene transduction into HLOs was achieved via a transient 2-dimensional culture during viral infection. This study provides an invaluable platform for utilizing HLOs in various research fields, such as molecular biology, pharmacology, toxicology, and regenerative medicine.

TLR7/8 stress response drives histiocytosis in SLC29A3 disorders.

Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs. However, little is known about the mechanism by which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3-/- mice was shown to depend on Toll-like receptor 7 (TLR7), which senses a combination of nucleosides and oligoribonucleotides (ORNs). TLR7 increased phagocyte numbers by driving the proliferation of Ly6Chi immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3-/- mice. Downstream of TLR7, FcRγ and DAP10 were required for monocyte proliferation. Histiocytosis is accompanied by inflammation in SLC29A3 disorders. However, TLR7 in nucleoside-laden splenic monocytes failed to activate inflammatory responses. Enhanced production of proinflammatory cytokines was observed only after stimulation with ssRNAs, which would increase lysosomal ORNs. Patient-derived monocytes harboring the G208R SLC29A3 mutation showed enhanced survival and proliferation in a TLR8-antagonist-sensitive manner. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies.

The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spätzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, we revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, we found that the upstream SPs Hayan and Psh contributes to the sterile activation of the Toll pathway. While SPE/MP1 double mutants show more impairment of Toll activation upon infection than SPE single mutants, Toll activation is not eliminated in these apoptosis-deficient flies. This suggests that Hayan and Psh sense necrotic damage, inducing Spz cleavage by SPs other than SPE and MP1. Furthermore, hydrogen peroxide, a representative damage-associated molecule, activates the Psh-Spz cascade in S2 cells overexpressing Psh. Considering that reactive oxygen species (ROS) were detected in apoptosis-deficient wings, our findings highlight the importance of ROS as signaling molecules that induce the activation of SPs such as Psh in response to damage.

Drug cytotoxicity screening using human intestinal organoids propagated with extensive cost-reduction strategies.

Organoids are regarded as physiologically relevant cell models and useful for compound screening for drug development; however, their applications are currently limited because of the high cost of their culture. We previously succeeded in reducing the cost of human intestinal organoid culture using conditioned medium (CM) of L cells co-expressing Wnt3a, R-spondin1, and Noggin. Here, we further reduced the cost by replacing recombinant hepatocyte growth factor with CM. Moreover, we showed that embedding organoids in collagen gel, a more inexpensive matrix than Matrigel, maintains organoid proliferation and marker gene expression similarly when using Matrigel. The combination of these replacements also enabled the organoid-oriented monolayer cell culture. Furthermore, screening thousands of compounds using organoids expanded with the refined method identified several compounds with more selective cytotoxicity against organoid-derived cells than Caco-2 cells. The mechanism of action of one of these compounds, YC-1, was further elucidated. We showed that YC-1 induces apoptosis through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway, the mechanism of which was distinct from cell death caused by other hit compounds. Our cost-cutting methodology enables large-scale intestinal organoid culture and subsequent compound screening, which could expand the application of intestinal organoids in various research fields.

Hydroxylation site-specific and production-dependent effects of endogenous oxysterols on cholesterol homeostasis: Implications for SREBP-2 and LXR.

The cholesterol metabolites, oxysterols, play central roles in cholesterol feedback control. They modulate the activity of two master transcription factors that control cholesterol homeostatic responses, sterol regulatory element-binding protein-2 (SREBP-2) and liver X receptor (LXR). Although the role of exogenous oxysterols in regulating these transcription factors has been well established, whether endogenously synthesized oxysterols similarly control both SREBP-2 and LXR remains poorly explored. Here, we carefully validate the role of oxysterols enzymatically synthesized within cells in cholesterol homeostatic responses. We first show that SREBP-2 responds more sensitively to exogenous oxysterols than LXR in Chinese hamster ovary cells and rat primary hepatocytes. We then show that 25-hydroxycholesterol (25-HC), 27-hydroxycholesterol, and 24S-hydroxycholesterol endogenously synthesized by CH25H, CYP27A1, and CYP46A1, respectively, suppress SREBP-2 activity at different degrees by stabilizing Insig (insulin-induced gene) proteins, whereas 7α-hydroxycholesterol has little impact on SREBP-2. These results demonstrate the role of site-specific hydroxylation of endogenous oxysterols. In contrast, the expression of CH25H, CYP46A1, CYP27A1, or CYP7A1 fails to induce LXR target gene expression. We also show the 25-HC production-dependent suppression of SREBP-2 using a tetracycline-inducible CH25H expression system. To induce 25-HC production physiologically, murine macrophages are stimulated with a Toll-like receptor 4 ligand, and its effect on SREBP-2 and LXR is examined. The results also suggest that de novo synthesis of 25-HC preferentially regulates SREBP-2 activity. Finally, we quantitatively determine the specificity of the four cholesterol hydroxylases in living cells. Based on our current findings, we conclude that endogenous side-chain oxysterols primarily regulate the activity of SREBP-2, not LXR.

Pathophysiological levels of GDF11 activate Smad2/Smad3 signaling and induce muscle atrophy in human iPSC-derived myocytes.

Skeletal muscle mass is negatively regulated by several TGF-ß superfamily members. Myostatin (MSTN) is the most prominent negative regulator of muscle mass. Recent studies show that in addition to MSTN, GDF11, which shares a high sequence identity with MSTN, induces muscle atrophy in vitro and in vivo at supraphysiological levels, whereas controversy regarding its roles exists. Furthermore, higher circulating GDF11 levels associate with frailty in humans. On the other hand, little is known about the effect of pathophysiological levels of GDF11 on muscle atrophy. Here we seek to determine whether pathophysiological levels of GDF11 are sufficient to activate Smad2/Smad3 signaling and induce muscle atrophy using human iPSC-derived myocytes (hiPSC myocytes). We first show that incubating hiPSC myocytes with pathophysiological concentrations of GDF11 significantly reduces myocyte diameters. We next demonstrate that pathophysiological levels of GDF11 are sufficient to activate Smad2/3 signaling. Finally, we show that pathophysiological levels of GDF11 are capable of inducing the expression of Atrogin-1, an atrophy-promoting E3 ubiquitin ligase and that FOXO1 blockage reverses the GDF11-induced Atrogin-1 expression and atrophic phenotype. Collectively, our results suggest that GDF11 induces skeletal muscle atrophy at the pathophysiological levels through the GDF11-FOXO1 axis.

Lactic acid bacteria-derived γ-linolenic acid metabolites are PPARδ ligands that reduce lipid accumulation in human intestinal organoids.

Gut microbiota regulate physiological functions in various hosts, such as energy metabolism and immunity. Lactic acid bacteria, including Lactobacillus plantarum, have a specific polyunsaturated fatty acid saturation metabolism that generates multiple fatty acid species, such as hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and trans-fatty acids. How these bacterial metabolites impact host physiology is not fully understood. Here, we investigated the ligand activity of lactic acid bacteria-produced fatty acids in relation to nuclear hormone receptors expressed in the small intestine. Our reporter assays revealed two bacterial metabolites of γ-linolenic acid (GLA), 13-hydroxy-cis-6,cis-9-octadecadienoic acid (γHYD), and 13-oxo-cis-6,cis-9-octadecadienoic acid (γKetoD) activated peroxisome proliferator-activated receptor delta (PPARδ) more potently than GLA. We demonstrate that both γHYD and γKetoD bound directly to the ligand-binding domain of human PPARδ. A docking simulation indicated that four polar residues (T289, H323, H449, and Y473) of PPARδ donate hydrogen bonds to these fatty acids. Interestingly, T289 does not donate a hydrogen bond to GLA, suggesting that bacterial modification of GLA introducing hydroxy and oxo group determines ligand selectivity. In human intestinal organoids, we determined γHYD and γKetoD increased the expression of PPARδ target genes, enhanced fatty acid ß-oxidation, and reduced intracellular triglyceride accumulation. These findings suggest that γHYD and γKetoD, which gut lactic acid bacteria could generate, are naturally occurring PPARδ ligands in the intestinal tract and may improve lipid metabolism in the human intestine.

Nobiletin enhances plasma Interleukin-6 and C-X-C motif chemokine ligand 1 levels that are increased by treadmill running.

Exercise increases the muscular secretion of Interleukin-6 (IL-6), which is partially regulated by ß2-adrenergic receptor signaling. Nobiletin is a polymethoxyflavone (PMF) found in citrus fruits that induces the secretion of IL-6 from C2C12 myotubes, but it remains unclear whether nobiletin promotes IL-6 secretion during exercise. The aim of this study was to clarify the effects of nobiletin on IL-6 secretion during exercise. Nobiletin and epinephrine were found to synergistically increase IL-6 secretion from differentiated C2C12 cells, which was suppressed by the inhibition of adenylyl cyclase (AC) or protein kinase A (PKA). Treadmill running for 60 min increased plasma levels of IL-6, epinephrine, and norepinephrine in rats. Nobiletin (5 mg/kg) orally administered 30 min before running increased plasma IL-6 levels further, although it did not increase plasma epinephrine and norepinephrine. In a similar manner to IL-6, nobiletin and epinephrine synergistically increased the secretion of C-X-C motif chemokine ligand 1 (CXCL-1) from C2C12 cells, or the increase in plasma CXCL-1 was enhanced by nobiletin after treadmill running of rats. Our results suggest that nobiletin promotes IL-6 and CXCL-1 secretion from skeletal muscle by synergistic enhancement of the PKA pathway in ß2-adrenergic receptor signaling.

Organoid-derived intestinal epithelial cells are a suitable model for preclinical toxicology and pharmacokinetic studies.

Intestinal organoids are physiologically relevant tools used for cellular models. However, the suitability of organoids to examine biological functions over existing established cell lines lacks sufficient evidence. Cytochrome P450 3A4 (CYP3A4) induction by pregnane X receptor ligands, glucose uptake via sodium/glucose cotransporter 1, and microsomal triglyceride transfer protein-dependent ApoB-48 secretion, which are critical for human intestinal metabolism, were observed in organoid-derived two-dimensional cells but little in Caco-2 cells. CYP3A4 induction evaluation involved a simplified method of establishing organoids that constitutively expressed a reporter gene. Compound screening identified several anticancer drugs with selective activities toward Caco-2 cells, highlighting their characteristics as cancer cells. Another compound screening revealed a decline in N-(4-hydroxyphenyl)retinamide cytotoxicity upon rifampicin treatment in organoid-derived cells, under CYP3A4-induced conditions. This study shows that organoid-derived intestinal epithelial cells (IECs) possess similar physiological properties as intestinal epithelium and can serve as tools for enhancing the prediction of biological activity in humans.

Sesamin and Hepatic Metabolites Derived from Sesamin and Episesamin Antagonize Farnesoid X Receptor and Reduce the Expression of Gluconeogenesis-Related Genes.

Sesamin and episesamin are the main lignans found in refined sesame oil and have been reported to exert various health benefits. However, the health benefits of these lignans and their molecular mechanisms have not been fully understood. This study evaluated the effects of sesamin, episesamin, and their metabolites on the nuclear bile acid receptor, farnesoid X receptor (FXR, NR1H4), which regulate gene expression involved in bile acid metabolism and gluconeogenesis. By using two different cell-based luciferase reporter assay systems, we found that sesamin, sesamin metabolites, and some episesamin metabolites inhibited FXR activation driven by a bile acid and a synthesized agonist, and it is suggested that these compounds exert their antagonist activity by competing with the FXR agonists on the ligand-binding domain. Sesamin and its major metabolite SC-1 suppressed the expression of several gluconeogenesis-related genes governed by FXR in HepG2 cells but did not affect the expression level of CYP7A1, the rate-limiting enzyme for bile acid synthesis. Dietary sesamin supplementation (AIN-93G supplemented with 0.5% sesamin) led to the decreased hepatic expression of several gluconeogenesis-related genes and reduced blood glucose levels in mice, without adverse effects on bile acid metabolism. These results shed light on the health benefits of taking sesamin and episesamin.

Skeletal muscle releases extracellular vesicles with distinct protein and microRNA signatures that function in the muscle microenvironment.

Extracellular vesicles (EVs) contain various regulatory molecules and mediate intercellular communications. Although EVs are secreted from various cell types, including skeletal muscle cells, and are present in the blood, their identity is poorly characterized in vivo, limiting the identification of their origin in the blood. Since skeletal muscle is the largest organ in the body, it could substantially contribute to circulating EVs as their source. However, due to the lack of defined markers that distinguish skeletal muscle-derived EVs (SkM-EVs) from others, whether skeletal muscle releases EVs in vivo and how much SkM-EVs account for plasma EVs remain poorly understood. In this work, we perform quantitative proteomic analyses on EVs released from C2C12 cells and human iPS cell-derived myocytes and identify potential marker proteins that mark SkM-EVs. These markers we identified apply to in vivo tracking of SkM-EVs. The results show that skeletal muscle makes only a subtle contribution to plasma EVs as their source in both control and exercise conditions in mice. On the other hand, we demonstrate that SkM-EVs are concentrated in the skeletal muscle interstitium. Furthermore, we show that interstitium EVs are highly enriched with the muscle-specific miRNAs and repress the expression of the paired box transcription factor Pax7, a master regulator for myogenesis. Taken together, our findings confirm previous studies showing that skeletal muscle cells release exosome-like EVs with specific protein and miRNA profiles in vivo and suggest that SkM-EVs mainly play a role within the muscle microenvironment where they accumulate.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids.

Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

Insulin-induced genes INSIG1 and INSIG2 mediate oxysterol-dependent activation of the PERK-eIF2α-ATF4 axis.

Insulin-induced genes (INSIGs) encode endoplasmic reticulum-resident proteins that regulate intracellular cholesterol metabolism. Oxysterols are oxygenated derivatives of cholesterol, some of which orchestrate lipid metabolism via interaction with INSIGs. Recently, it was reported that expression of activating transcription factor-4 (ATF4) was induced by certain oxysterols; the precise of mechanism is unclear. Herein, we show that INSIGs mediate ATF4 upregulation upon interaction with oxysterol. Oxysterols that possess a high affinity for INSIG, such as 27- and 25-hydroxycholesterol (25HC), markedly induced the increase of ATF4 protein when compared with other oxysterols. In addition, ATF4 upregulation by these oxysterols was attenuated in INSIG1/2-deficient Chinese hamster ovary cells and recovered by either INSIG1 or INSIG2 rescue. Mechanistic studies revealed that the binding of 25HC to INSIG is critical for increased ATF4 protein via activation of protein kinase RNA-activated-like ER kinase and eukaryotic translation initiation factor 2α. Knockout of INSIG1 or INSIG2 in human hepatoma Huh7 cells attenuated ATF4 protein upregulation, indicating that only one of the endogenous INSIGs, unlike overexpression of intrinsic INSIG1 or INSIG2, was insufficient for ATF4 induction. Furthermore, ATF4 proactively upregulated the cell death-inducible gene expression, such as Chop, Chac1, and Trb3, thereby markedly reducing cell viability with 25HC. These findings support a model whereby that INSIGs sense an increase in oxysterol in the endoplasmic reticulum and induce an increase of ATF4 protein via the protein kinase RNA-activated-like ER kinase-eukaryotic translation initiation factor 2α pathway, thereby promoting cell death.

Orange peel extract reduces the inflammatory state of skeletal muscle after downhill running via an increase in IL-1RA.

It has been reported that orange peel extract (OPE) and the 4 major polymethoxyflavones (PMFs) in OPE have a protective effect against downhill running (DR)-induced skeletal muscle inflammation. However, the mechanism is not well understood. We investigated the potential of OPE and PMF compounds for increasing anti-inflammatory cytokine levels. The plasma interleukin-1 receptor antagonist (IL-1RA) level was increased 1 and 8 h after OPE administration in rats. Nobiletin induced the secretion of IL-1RA from C2C12 myotubes. In the inflammatory state of skeletal muscle after DR, OPE administration reduced nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) expression, NF-κB-DNA binding, and monocyte chemotactic protein-1 mRNA levels, but these effects were all abrogated by the intravenous administration of IL-1RA neutralizing antibody. These results indicated that OPE reduces skeletal muscle inflammatory state after DR via an increase in IL-1RA, and that IL-1 receptor signaling is important for skeletal muscle inflammation after DR.

Polymethoxyflavones in orange peel extract prevent skeletal muscle damage induced by eccentric exercise in rats.

Polymethoxyflavones (PMFs) contained in the peel of citrus fruits have anti-inflammatory, anticancer, and antidepressant effects. However, their effects on skeletal muscle are unknown. We investigated whether PMFs could prevent skeletal muscle damage induced by eccentric exercise in rats. Downhill running for 90 min increased the levels of the inflammatory cytokines, monocyte chemotactic protein-1 (MCP-1), and interleukin-1ß (IL-1ß) in skeletal muscles, especially in vastus lateralis, and the plasma creatine kinase levels. These increases were attenuated by a single oral administration of orange peel extract (OPE) 30 min before downhill running. A mixture of nobiletin, sinensetin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin, which are the major PMFs of OPE, also showed similar effects on muscle damage. These results suggest that OPE has a protective effect against eccentric exercise-induced skeletal muscle damage, and that the effects may be attributed to the 4 major PMFs.

Muscle-specific TGR5 overexpression improves glucose clearance in glucose-intolerant mice.

TGR5, a G protein-coupled bile acid receptor, is expressed in various tissues and regulates several physiological processes. In the skeletal muscle, TGR5 activation is known to induce muscle hypertrophy; however, the effects on glucose and lipid metabolism are not well understood, despite the fact that the skeletal muscle plays a major role in energy metabolism. Here, we demonstrate that skeletal muscle-specific TGR5 transgenic (Tg) mice exhibit increased glucose utilization, without altering the expression of major genes related to glucose and lipid metabolism. Metabolite profiling analysis by capillary electrophoresis time-of-flight mass spectrometry showed that glycolytic flux was activated in the skeletal muscle of Tg mice, leading to an increase in glucose utilization. Upon long-term, high-fat diet challenge, blood glucose clearance was improved in Tg mice without an accompanying increase in insulin sensitivity in skeletal muscle and a reduction of body weight. Moreover, Tg mice showed improved age-associated glucose intolerance. These results strongly suggest that TGR5 ameliorated glucose metabolism disorder that is caused by diet-induced obesity and aging by enhancing the glucose metabolic capacity of the skeletal muscle. Our study demonstrates that TGR5 activation in the skeletal muscle is effective in improving glucose metabolism and may be beneficial in developing a novel strategy for the prevention or treatment of hyperglycemia.

Cryo-EM structure of the highly atypical cytoplasmic ribosome of Euglena gracilis.

Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.

Functional effect of nobiletin as a food-derived allosteric modulator of mouse CRFR2ß in skeletal muscle.

Activation of corticotropin-releasing factor receptor 2ß (CRFR2ß) results in increased skeletal muscle mass and the prevention of muscle atrophy. Using a luciferase reporter assay, we screened 357 functional food factors that activate CRFR2ß and, subsequently, confirmed that nobiletin (NBT) increases CRFR2ß activity. Additionally, we found that NBT augments the activity of the endogenous peptide ligand urocortin 2 (Ucn2) in a concentration-dependent manner. Computational simulation of CRFR2ß confirmed that transmembrane domains (TMs) 1 and 2 are important for the synergistic activity of NBT and also identified important amino acids in these domains. Finally, we demonstrated that a co-administration of Ucn2 and NBT increases the hypertrophic signal in mouse skeletal muscle. These observations demonstrate that NBT can activate CRFR2ß and amplify the agonistic activity of Ucn2 and that such food-derived molecules have the potential to enhance endogenous G protein-coupled receptor ligand activities and contribute to the maintenance of skeletal muscle mass and function.

Direct Determination of Pseudouridine in RNA by Mass Spectrometry Coupled with Stable Isotope Labeling.

Pseudouridine (Ψ) is the only "mass-silent" nucleoside produced by post-transcriptional RNA modification. We developed a mass spectrometry (MS)-based technique coupled with in vivo deuterium (D) labeling of uridines for direct determination of Ψs in cellular RNA and applied it to the comprehensive analysis of post-transcriptional modifications in human ribosomal RNAs. The method utilizes human TK6/mouse FM3A cells deficient in uridine monophosphate synthase using a CRISPR-Cas9 technique to turn off de novo uridine synthesis and fully labels uridines with D at uracil positions 5 and 6 by cultivating the cells in a medium containing uridine-5,6-D2. The pseudouridylation reaction in those cells results in the exchange of the D at the C5 of uracil with hydrogen from solvent, which produces a -1 Da mass shift, thus allowing MS-based determination of RNA Ψs. We present here the experimental details of this method and show that it allows the identification of all Ψs in human major nuclear and nucleolar RNAs, including several previously unknown Ψs. Because the method allows direct determination of Ψs at the femtomole level of RNA, it will serve as a useful tool for structure/function studies of a wide variety of noncoding RNAs.