Objective The vagus nerve provides a direct line of communication between the gut and the brain for proper regulation of energy balance and glucose homeostasis. Short-chain fatty acids (SCFAs) produced via gut microbiota fermentation of dietary fiber have been proposed to regulate host metabolism and feeding behavior via the vagus nerve, but the molecular mechanisms have not yet been elucidated. We sought to identify the G-protein-coupled receptors within vagal neurons that mediate the physiological and therapeutic benefits of SCFAs. Methods SCFA, particularly propionate, signaling occurs via free fatty acid receptor 3 (FFAR3), that we found expressed in vagal sensory neurons innervating throughout the gut. The lack of cell-specific animal models has impeded our understanding of gut/brain communication; therefore, we generated a mouse model for cre-recombinase-driven deletion of Ffar3 . We comprehensively characterized the feeding behavior of control and vagal-FFAR3 knockout (KO) mice in response to various conditions including fasting/refeeding, western diet (WD) feeding, and propionate supplementation. We also utilized ex vivo organotypic vagal cultures to investigate the signaling pathways downstream of propionate FFAR3 activation. Results Vagal-FFAR3KO led to increased meal size in males and females, and increased food intake during fasting/refeeding and WD challenges. In addition, the anorectic effect of propionate supplementation was lost in vagal-FFAR3KO mice. Sequencing approaches combining ex vivo and in vivo experiments revealed that the cross-talk of FFAR3 signaling with cholecystokinin (CCK) and leptin receptor pathways leads to alterations in food intake. Conclusion Altogether, our data demonstrate that FFAR3 expressed in vagal neurons regulates feeding behavior and mediates propionate-induced decrease in food intake.

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Obesity is an epidemic, and it is characterized by a state of low-grade systemic inflammation A key component of inflammation is the activation of inflammasomes, multiprotein complexes that form in response to danger signals and that lead to activation of caspase-1. Previous studies have found that a Westernized diet induces activation of inflammasomes and production of inflammatory cytokines. Gut microbiota metabolites, including the short-chain fatty acid butyrate, have received increased attention as underlying some obesogenic features, but the mechanisms of action by which butyrate influences inflammation in obesity remain unclear. We engineered a caspase-1 reporter mouse model to measure spatiotemporal dynamics of inflammation in obese mice. Concurrent with increased capsase-1 activation in vivo, we detected stronger biosensor signal in white adipose and heart tissues of obese mice ex vivo and observed that a short-term butyrate treatment affected some but not all the inflammatory responses induced by Western diet. Through characterization of inflammatory responses and computational analyses, we identified tissue- and sex-specific caspase-1 activation patterns and inflammatory phenotypes in obese mice, offering new mechanistic insights underlying the dynamics of inflammation.

Artemisinin (ART), a common anti‐malarial drug, and its analogs are also useful as repurposed anti‐cancer drugs, but their properties are not fully elucidated. We previously synthesized novel trioxane (DMR) and dioxazanine (HSM) ART analogs and showed that: a. at low doses (5–10 μM), HSM > than DMR in inducing apoptosis in human colon and lung cancer cells, but not in normal lung cells; b. The actions involved reactive oxygen species (ROS) (Faseb J, ′18:32, 616.2). TfR expression is increased in cancer vs. normal cells. Thus, we hypothesize that, in cancer cells, TfR increases [Fe2+]i which acts on the endoperoxide to produce ROS and induce apoptosis. We examined the mechanism of action of DMR/HSM in inducing apoptosis in human breast, lung, and colon cancer cell lines as compared to normal cell lines. To increase the efficacy of the drug, we also designed and synthesized a 3rd novel analog with dual dioxazanine pharmacophores, PMW.Confluent normal (Breast: MCF10A, Lung: A549) and cancer (Breast: MCF7, Colon: T84, Lung: A549) cells were treated (18 H) with 1–50μM of DMR, HSM or PMW±Deferoxamine (DFO, iron chelator; 1–25 μM). Cells were stained with FITC‐Annexin V (apoptosis), propidium iodide (cell death) or CellRox Green (mitochondrial/nuclear ROS), imaged and quantified by flow cytometry and/or microscopy (Image J). Normal and cancer cell lysates (30 μg) were subjected to SDS‐PAGE and immunoblotting with polyclonal TfR antibody or anti‐GAPDH (control).As in other cancer cells, both DMR and HSM (5 – 50μM) induced apoptosis in breast cancer, but not normal, cells. In contrast to A549 lung cells, where HSM was more effective than DMR, in MCF7 breast cells, 50μM DMR was more efficient (~40%) in inducing apoptosis than HSM (10%). This dictates the development of novel tissue‐specific analogs. The new analog, PMW (1 – 50μM, 18H) did not induce apoptosis in the cancer cells. The larger size of PMW may have slowed entry, suggesting longer incubations.Image J analysis (mean pixel intensity; 18 H) of CellRox+ lung cells (n=3) showed that HSM dose‐dependently increased ROS in cancer A549: (DMSO: 1.5±0.2; HSM, 5 μM: 19±2; 10 μM: 48±4; 50 μM: 59±2); but not normal BEAS2B: (DMSO: 1.0±0.3; HSM, 5 μM: 1.8±1; 10 μM: 2.2±0.4; 50 μM: 2.9±2). Similarly, in breast cancer cells (n=3), DMR dose‐dependently increased ROS in MCF7 (DMSO: 1.0±0.3; DMR, 5 μM: 12±2; 10 μM: 51±5; 50 μM: 42±3); but not in MCF10A (DMSO: 1.0±0.2; DMR, 5 μM: 1.6±1; 10 μM: 3.1±1; 50 μM: 2.5±1).Immunoblotting detected a distinct 94 kDa TfR protein in colon, lung, and breast cancer, but not normal cells. The effect of DMR/HSM±DFO on apoptosis was examined to study the role of TfR. Albeit showing an inhibitory trend, DFO did not significantly alter ART analogs actions (% Annexin V+ cells,18 H: A549: HSM,10μM: 46±4; HSM+5μM DFO: 38±6; HSM +10μM DFO: 33±9; MCF7: DMR,10μM: 36±4; DMR+5μM DFO: 31±7; DMR+10μM DFO: 29±5; n≥3).Synthesizing novel ART‐analogs with improved pharmacokinetics to specifically target cancer cells and elucidating their mechanism of action will help develop new alternatives to treat cancer.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS; Ben U Funds to JS and DMR; UIC Funds to MCR; APS‐STRIDE to UD; APS‐UGSRF to MHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dysfunction of mucosal immune response and tight junctions (TJ) play an important role in the pathogenesis of inflammatory bowel diseases (IBD) and diarrhea. We reported that excess BA, namely chenodeoxycholic acid (CDCA; 500μM) induced PiC release and altered T84 TJ by increasing ROS. The effect on leak function of TJ was attenuated by CDCA's derivative, lithocholic acid (LCA, 50 μM) and ROS inhibitors (Physiol Rep, ′17, 5: e13294). In patients with IBD, PiC upregulate inducible nitric oxide synthase (iNOS). Thus, we hypothesize a role for RNS in mucosal damage and studied the involvement of oxidative/nitrosative stress in BA‐induced TJ dysfunction and cytokine release in T84 cells.We previously described the synthesis of fluorescein amine‐tagged CDCA (CDCA‐FA) and its use to track transepithelial (TE) BA movement. Confluent T84 cells (TE Resistance; TER >1000Ωcm2) were treated apically with DMSO, 500μM CDCA‐FA, 50μM LCA, CDCA+LCA, ± PiC ([ng/ml]: TNFα[10]+IL‐1β[10]+IFNγ[30]), ± 50μM L‐NAME ( L‐NG‐Nitroarginine methyl ester, NOS inhibitor), ± 1mM NAC, (N‐acetyl cysteine, ROS scavenger), for 0.5–18 H. [NO2/NO3] was measured by the Griess assay. We examined the role of RNS/ROS in BA action by studying the effect of BAs±L‐NAME± NAC on: a. Apoptosis (Annexin V, Flow cytometry); b. Paracellular permeability (Pore function as TER; Leak function as TE CDCA‐FA movement); and c. IL‐8 release (ELISA).CDCA (18 H), but not LCA, increased [NO2/NO3] 3‐fold and this was enhanced by PiC (μmol/mg protein; DMSO: 12±1; CDCA: 36±1; LCA: 16± 4, PiC: 18±1; PiC+CDCA: 55±2, n=3). LCA decreased CDCA±PiC‐induced [NO2/NO3] (CDCA+LCA: 25±1; PiC+LCA: 13±2, PiC+CDCA+LCA: 27±4; p<0.05). Inhibiting RNS did not alter CDCA‐induced apoptosis. In pore function, L‐NAME reduced the initial rate of CDCA‐FA‐induced decrease in TER (Ωcm2/sec; 1H CDCA‐FA: 12±1; CDCA‐FA+L‐NAME: 7±1, n>3; p<0.05 ), with no statistical difference at 4 and 18H. L‐NAME altered leak function, reducing CDCA‐FA flux by ~30% at 18 H, (Apparent permeability: Papp ×10−9 cm/sec: CDCA‐FA: 57±2; CDCA‐FA+L‐NAME: 30±1; p<0.05, n=3). LCA decreased CDCA‐FA flux by ~58%, and LCA+L‐NAME completely attenuated it (CDCA‐FA+LCA: 24±2; CDCA‐FA+LCA+L‐NAME: 3±1; p<0.05, n>3). NAC+LCA caused only a ~85% decrease in CDCA‐FA flux (CDCA‐FA+LCA+NAC: 8±2).Like CDCA, CDCA‐FA±PiC stimulated IL‐8 release, which was decreased by LCA. Inhibiting RNS, but not ROS, caused ~40% reduction in CDCA‐induced IL‐8 release (ng/ml; CDCA: 4.6±0.5; CDCA+L‐NAME: 2.7±0.1; CDCA+NAC: 4.2±0.3; n=3), suggesting a role for NO in BA‐induced inflammatory process.We demonstrate a novel role for RNS in CDCA‐induced TJ dysfunction in T84 cells. Inhibiting RNS in the presence of LCA reverses CDCA action on leak function and reduces PiC release. Equally important, its roles overlap with but are distinct from ROS. Understanding the role of ROS/RNS in BA action can lead to novel therapeutic strategies for IBD.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS and Ben U Funds to JS and DMR; UIC Funds to MCR; APS‐STRIDE National Heart, Lung and Blood Institute (Grant #1 R25 HL115473‐01) to UD; APS‐UGSRF to MHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.