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Farnesoid-x-receptor (FXR) agonists, currently trialed in patients with non-alcoholic steatosis (NAFLD), worsen the pro-atherogenic lipid profile and might require a comedication with statin. Here we report that mice feed a high fat/high cholesterol diet (HFD) are protected from developing a pro-atherogenic lipid profile because their ability to dispose cholesterol through bile acids. This protective mechanism is mediated by suppression of FXR signaling in the liver by muricholic acids (MCAs) generated in mice from chenodeoxycholic acid (CDCA). In contrast to CDCA, MCAs are FXR antagonists and promote a CYP7A1-dependent increase of bile acids synthesis. In mice feed a HFD, the treatment with obeticholic acid, a clinical stage FXR agonist, failed to improve the liver histopathology while reduced Cyp7a1 and Cyp8b1 genes expression and bile acids synthesis and excretion. In contrast, treating mice with atorvastatin mitigated liver and vascular injury caused by the HFD while increased the bile acids synthesis and excretion. Atorvastatin increased the percentage of 7α-dehydroxylase expressing bacteria in the intestine promoting the formation of deoxycholic acid and litocholic acid, two GPBAR1 agonists, along with the expression of GPBAR1-regulated genes in the white adipose tissue and colon. In conclusion, present results highlight the central role of bile acids in regulating lipid and cholesterol metabolism in response to atorvastatin and provide explanations for limited efficacy of FXR agonists in the treatment of NAFLD., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Autophagy is a highly conserved catabolic process activated by fasting and caloric restriction. FXR, a receptor for primary bile acids, reverses the activity of cAMP-response element binding protein (CREB) on autophagy-related genes (Atg)s and terminates autophagy in the fed state. GPBAR1, a receptor for secondary bile acids, exerts its genomic effects via cAMP-CREB pathway. By genetic and pharmacological approaches, we have obtained evidence that GPBAR1 functions as a positive modulator of autophagy in liver and white adipose tissue (WAT) in fasting. Mechanistically, we found that Gpbar1 -/- mice lack the expression of Cyp2c70 a gene essential for generation of muricholic acids which are FXR antagonists, and have an FXR-biased bile acid pool. Because FXR represses autophagy, Gpbar1 -/- mice show a defective regulation of autophagy in fasting. BAR501, a selective GPBAR1 agonist, induces autophagy in fed mice. Defective regulation of autophagy in Gpbar1 -/- could be reversed by FXR antagonism, while repression of autophagy by feeding was partially abrogated by FXR gene ablation, and FXR activation repressed Atgs in the fast state. BAR501 reversed the negative regulatory effects of feeding and FXR agonism on autophagy and promoted the recruitment of CREB to a CRE on the LC3 promoter. In mice exposed to chronic high caloric intake, GPBAR1 agonism ameliorated insulin sensitivity and induced Atgs expression in the liver and WAT. In summary, GPBAR1 is required for positive regulation of autophagy in fasting and its ligands reverse the repressive effects exerted on liver and WAT autophagy flow by FXR in fed., (© 2020 Federation of American Societies for Experimental Biology.)

Nonalcoholic steatohepatitis (NASH) is associated with an increased risk of developing cardiovascular complications and mortality, suggesting that treatment of NASH might benefit from combined approaches that target the liver and the cardiovascular components of NASH. Using genetic and pharmacologic approaches, we show that G protein-coupled bile acid-activated receptor 1 (GPBAR1) agonism reverses liver and vascular damage in mouse models of NASH. NASH is associated with accelerated vascular inflammation representing an independent risk factor for development of cardiovascular diseases and cardiovascular-related mortality. GPBAR1, also known as TGR5, is a G protein-coupled receptor for secondary bile acids that reduces inflammation and promotes energy expenditure. Using genetic and pharmacologic approaches, we investigated whether GPBAR1 agonism by 6β-ethyl-3α,7β-dihydroxy-5β-cholan-24-ol (BAR501) reverses liver and vascular damage induced by exposure to a diet enriched in fat and fructose (HFD-F). Treating HFD-F mice with BAR501 reversed liver injury and promoted the browning of white adipose tissue in a Gpbar1-dependent manner. Feeding HFD-F resulted in vascular damage, as shown by the increased aorta intima-media thickness and increased expression of inflammatory genes (IL-6,TNF-α, iNOS, and F4/80) and adhesion molecules (VCAM, intercellular adhesion molecule-1, and endothelial selectin) in the aorta, while reducing the expression of genes involved in NO and hydrogen sulfide generation, severely altering vasomotor activities of aortic rings in an ex vivo assay. BAR501 reversed this pattern in a Gpbar1-dependent manner, highlighting a potential role for GPBAR1 agonism in treating the liver and vascular component of NASH.-Carino, A., Marchianò, S., Biagioli, M., Bucci, M., Vellecco, V., Brancaleone, V., Fiorucci, C., Zampella, A., Monti, M. C., Distrutti, E., Fiorucci, S. Agonism for the bile acid receptor GPBAR1 reverses liver and vascular damage in a mouse model of steatohepatitis.

The glucocorticoid receptor (GR) is a master gene orchestrating the activation of gluconeogenic genes in the liver in response to food withdrawal. Mechanisms of GR regulation by other nuclear receptors, however, are poorly defined. Here, we report that the farnesoid X receptor (FXR), a bile acid sensor, activates gluconeogenic pathways in the liver and regulates GR expression and activity. FXR-null mice are hypoglycemic in the unfed state and exhibit both a reduced hepatic production of glucose in response to the pyruvate challenge and a decreased expression of two rate-limiting enzymes involved in gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase), along with blunted liver expression of GR. Treating wild-type mice with a semisynthetic FXR ligand (6E-CDCA) increases the liver expression of GR, PEPCK, and G6Pase. This effect was lost in fed animals, as well as in FXR(-/-) mice. The human and mouse GR promoters contain a conserved FXR-responsive element (an ER-8 sequence) whose activation by FXR ligation leads to GR transcription. GR silencing by siRNA in vitro or its pharmacological antagonism in vivo with mifepristone reverses the effect of FXR activation on expression of gluconeogenic genes. These findings demonstrate that an FXR-GR pathway regulates the activation of hepatic gluconeogenesis in the transition from the unfed to the fed state.

Hydrogen sulfide (H2S) is increasingly recognized as an important signaling molecule in the cardiovascular and nervous systems. Recently, H2S donors were reported to induce neutrophil apoptosis and to suppress expression of some leukocyte and endothelial adhesion molecules. Using rats, we examined the possibility that H2S is an endogenous regulator of key inflammatory events at the leukocyte-endothelial interface. Via intravital microscopy, we observed that H2S donors (NaHS and Na2S) inhibited aspirin-induced leukocyte adherence in mesenteric venules (ED50 of 5.0 micromol/kg for Na2S), likely via activation of ATP-sensitive K+ (K(ATP)) channels. Inhibition of endogenous H2S synthesis elicited leukocyte adherence. Leukocyte infiltration in an air pouch model was also suppressed by H2S donors (NaHS, Lawesson's reagent, and N-acetylcysteine; ED50 of 42.7, 1.3, and 29.9 micromol/kg, respectively) and exacerbated by inhibition of endogenous H2S synthesis. Carrageenan-induced paw edema was suppressed by H2S donors (NaHS and Na2S; ED50s of 35 and 28 micromol/kg, respectively) to the same extent as by diclofenac and enhanced by an inhibitor of H2S synthesis. Suppression of edema formation by H2S donors was mimicked by a K(ATP) channel agonist and reversed by an antagonist of this channel. These results suggest that endogenous H2S is an important mediator of acute inflammation, acting at the leukocyte-endothelium interface. These findings have important implications for anti-inflammatory drug development.

In addition to inhibiting formation of prothrombotic eicosanoids, aspirin causes the acetylation of cyclooxygenase (COX)-2. The acetylated COX-2 remains active, and upon cell activation, initiates the generation of 15R-HETE, a lipid substrate for 5-lipoxygenase (LOX) leading to the formation of 15-epi-LXA4 (also termed "aspirin-triggered lipoxin," or ATL). Because ATL potently inhibits polymorphonuclear cell (PMN) function, we assessed the relative contribution of this lipid mediator in conjunction with another 5-LOX product, the leukotriene (LT)B4, to the pathogenesis of acute damage and gastric adaptation to aspirin. Data presented herein indicate that acute injury and gastric adaptation to aspirin is associated with ATL generation. Administration of COX inhibitors (celecoxib, indomethacin, ketoprofen) to aspirin-treated rats exacerbated acute injury and abolished adaptation to aspirin. Moreover, it inhibited ATL formation and caused a four- to fivefold increase in LTB4 synthesis. In contrast, licofelone, a COX/5-LOX inhibitor, did not exacerbate acute gastric injury nor did it interfere with gastric adaptation to aspirin. Although licofelone blocked ATL and LTB4 formation in aspirin-treated rats, it attenuated aspirin-induced gastric PMN margination. These findings indicate that the balance between the production of LTB4 and ATL modulates PMN recruitment/function and gastric mucosal responses to aspirin.

We investigated the effect of a nitric oxide (NO)-releasing derivative of aspirin (NCX-4016) on a mitochondria-dependent model of apoptosis in human umbilical endothelial cells (HUVEC). Exposure of HUVEC to staurosporine caused a progressive fall in mitochondrial membrane potential (DeltaPsi(m)) and apoptosis. Exposure to an NO donor, (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), caused an early (1-3h) hyperpolarization of DeltaPsi(m) and reduction of apoptosis that was followed (at 4-8 h) by an accelerated collapse of DeltaPsi(m) and cell death. In contrast, treatment with NCX-4016, but not with aspirin or a non-NO-releasing analogue of NCX-4016, protected HUVEC against the apoptotic actions of staurosporine for up to 8 h. Confocal microscopy demonstrated that although NCX-4016 released NO in subcellular compartments, DETA-NO caused a generalized increase in cytosolic fluorescence. Exposure to DETA-NO resulted in a rapid and profound inhibition of cell respiration (78.3 +/- 6.4%), whereas NCX-4016 caused a less pronounced reduction in oxygen consumption (43.5 +/- 5.3%). Staurosporine caused a time-dependent activation of proapoptotic caspases. NCX-4016 prevented this activation, whereas DETA-NO failed to inhibit caspase activity. In contrast to DETA-NO, NCX-4016 did not increase mitochondrial oxidative stress. These data demonstrated that NCX-4016 conveys NO directly inside endothelial cells and modulates mitochondrial function.