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Olfactory receptor 544 (Olfr544) is expressed in extra-nasal tissues and has been shown to reduce adiposity and shift fuel preference to fats. Here, we investigated whether Olfr544 regulates feeding behavior. Azelaic acid increased cholecystokinin (CCK) secretion ex vivo and in vivo by Olfr544 dependently. In a two-food choice study, oral administration of azelaic acid reduced high fat diet (HFD) intake and increased normal diet (ND) intake, which was abolished in Olfr544-knockout mice. In a two-bottle preference test, the oral administration of azelaic acid reduced the intake of linoleic acid solution while increasing the intake of control solution (xanthan gum) and the effect of azelaic acid disappeared in Olfr544-knockout mice. Intraperitoneal injection of proglumide, a CCK-1/2 receptor antagonist, negated the effect of azelaic acid in reducing fat preference. Collectively, our findings suggested that Olfr544 activation by azelaic acid reduced calorie-rich high-fat food intake mediated, at least in part, by CCK secretion.

Olfactory receptors are ectopically expressed in extra-nasal tissues. The gut is constantly exposed to high levels of odorants where ectopic olfactory receptors may play critical roles. Activation of ectopic olfactory receptor 544 (Olfr544) by azelaic acid (AzA), an Olfr544 ligand, reduces adiposity in mice fed a high-fat diet (HFD) by regulating fuel preference to fats. Herein, we investigated the novel function of Olfr544 in the gut. In GLUTag cells, AzA induces the cAMP-PKA-CREB signaling axis and increases the secretion of GLP-1, an enteroendocrine hormone with anti-obesity effects. In wild-type (WT) mice injected AzA, GLP-1 plasma levels were elevated. The induction of GLP-1 secretion was negated in cells with Olfr544 gene knockdown and in Olfr544-deficient mice. Gut microbiome analysis revealed that AzA increased the levels of Bacteroides acidifaciens and microbiota associated with antioxidant pathways. In fecal metabolomics analysis, the levels of succinate and trehalose, metabolites correlated with a lean phenotype, were elevated by AzA. The function of Olfr544 in gut inflammation, a key feature in obesity, was further investigated. In RNA sequencing analysis, AzA suppressed LPS-induced activation of inflammatory pathways and reduced TNF-α and IL-6 expression, thereby improving intestinal permeability. The effects of AzA on the gut metabolome, microbiome, and colon inflammation were abrogated in Olfr544-KO mice. These results collectively demonstrated that activation of Olfr544 by AzA in the gut exerts multiple effects by regulating GLP-1 secretion, gut microbiome and metabolites, and colonic inflammation in anti-obesogenic phenotypes and, thus, may be applied for obesity therapeutics.

The mouse olfactory receptor Olfr544 is expressed in several non-olfactory tissues and has been suggested as a functional receptor regulating different signaling pathways. However, the molecular interaction between Olfr544 and its natural ligand, azelaic acid (AzA), remains poorly characterized, primarily due to difficulties in the heterologous expression of the receptor protein on the cell membrane and lack of entire protein structure. In this report, we describe the molecular determinants of Olfr544 activation by AzA. N-terminal lucy-flag-rho tag ensured the heterologous expression of Olfr544 on the Hana3A cell surface. Molecular modeling and docking combined with mutational analysis identified amino acid residues in the Olfr544 for the interaction with AzA. Our data demonstrated that the Y109 residue in transmembrane helix 3 forms a hydrogen bond with AzA, which is crucial for the receptor-ligand interaction and activation. Y109 is required for the Olfr544 activation by AzA which, in turn, stimulates the Olfr544-dependent CREB-PGC-1α signaling axis and is followed by the induction of mitochondrial biogenesis in Olfr544 wild-type transfected Hana3A cells, but not in mock or Y109A mutant transfected cells. Collectively, these data indicated that a hydrogen bond between Y109 residue and AzA is a major determinant of the Olfr544-AzA interaction and activation. [ABSTRACT FROM AUTHOR]

[Display omitted] • Azelaic acid stimulated cholecystokinin secretion via activation of Olfr544. • Oral administration of azelaic acid decreased a high fat diet intake and increased normal diet intake, which was abrogated in Olfr544-deficient mice. • Proglumide, a CCK 1/2 receptor antagonist, abolished the effect of azelaic acid in reducing fat preference. • Our results suggest that activation of Olfr544 by azelaic acid reduced fat preference, mediated, at least in part, by CCK secretion. Olfactory receptor 544 (Olfr544) is expressed in extra-nasal tissues and has been shown to reduce adiposity and shift fuel preference to fats. Here, we investigated whether Olfr544 regulates feeding behavior. Azelaic acid increased cholecystokinin (CCK) secretion ex vivo and in vivo by Olfr544 dependently. In a two-food choice study, oral administration of azelaic acid reduced high fat diet (HFD) intake and increased normal diet (ND) intake, which was abolished in Olfr544-knockout mice. In a two-bottle preference test, the oral administration of azelaic acid reduced the intake of linoleic acid solution while increasing the intake of control solution (xanthan gum) and the effect of azelaic acid disappeared in Olfr544-knockout mice. Intraperitoneal injection of proglumide, a CCK-1/2 receptor antagonist, negated the effect of azelaic acid in reducing fat preference. Collectively, our findings suggested that Olfr544 activation by azelaic acid reduced calorie-rich high-fat food intake mediated, at least in part, by CCK secretion. [ABSTRACT FROM AUTHOR]

Olfactory receptors (ORs) are extensively expressed in olfactory as well as non-olfactory tissues. Although many OR transcripts are expressed in non-olfactory tissues, only a few studies demonstrate the functional role of ORs. Here, we verified that mouse pancreatic α-cells express potential OR-mediated downstream effectors. Moreover, high levels of mRNA for the olfactory receptors Olfr543, Olfr544, Olfr545, and Olfr1349 were expressed in α-cells as assessed using RNA-sequencing, microarray, and quantitative real-time RT-PCR analyses. Treatment with dicarboxylic acids (azelaic acid and sebacic acid) increased intracellular Ca 2+ mobilization in pancreatic α-cells. The azelaic acid-induced Ca 2+ response as well as glucagon secretion was concentration- and time-dependent manner. Olfr544 was expressed in α-cells, and the EC 50 value of azelaic acid to Olfr544 was 19.97 μM, whereas Olfr545 did not respond to azelaic acid. Our findings demonstrate that Olfr544 responds to azelaic acid to regulate glucagon secretion through Ca 2+ mobilization in α-cells of the mouse pancreatic islets, suggesting that Olfr544 may be an important therapeutic target for metabolic diseases. [ABSTRACT FROM AUTHOR]

Olfactory receptors are ectopically expressed in extra-nasal tissues. The gut is constantly exposed to high levels of odorants where ectopic olfactory receptors may play critical roles. Activation of ectopic olfactory receptor 544 (Olfr544) by azelaic acid (AzA), an Olfr544 ligand, reduces adiposity in mice fed a high-fat diet (HFD) by regulating fuel preference to fats. Herein, we investigated the novel function of Olfr544 in the gut. In GLUTag cells, AzA induces the cAMP-PKA-CREB signaling axis and increases the secretion of GLP-1, an enteroendocrine hormone with anti-obesity effects. In mice fed a HFD and orally administered AzA, GLP-1 plasma levels were elevated in mice. The induction of GLP-1 secretion was negated in cells with Olfr544 gene knockdown and in Olfr544-deficient mice. Gut microbiome analysis revealed that AzA increased the levels of Bacteroides acidifaciens and microbiota associated with antioxidant pathways. In fecal metabolomics analysis, the levels of succinate and trehalose, metabolites correlated with a lean phenotype, were elevated by AzA. The function of Olfr544 in gut inflammation, a key feature in obesity, was further investigated. In RNA sequencing analysis, AzA suppressed LPS-induced activation of inflammatory pathways and reduced TNF-α and IL-6 expression, thereby improving intestinal permeability. The effects of AzA on the gut metabolome, microbiome, and colon inflammation were abrogated in Olfr544-KO mice. These results collectively demonstrated that activation of Olfr544 by AzA in the gut exerts multiple effects by regulating GLP-1 secretion, gut microbiome and metabolites, and colonic inflammation in anti-obesogenic phenotypes and, thus, may be applied for obesity therapeutics.