Endocrine Abstracts

The Hydroxycarboxylic Acid Receptor 1 (HCAR1) is a Gαi/o-coupled GPCR known to be activated by both L- and D-lactate; metabolites produced by gut microbiota and anaerobic metabolism. Elevated D-lactate levels are indicative of dysbiosis and inflammatory bowel disease. As lactate can be transported into cells, it has the potential to activate HCAR1 at the plasma membrane and within intracellular locations resulting in diversified responses. Whether HCAR1 signalling is spatially regulated and if this influences gut barrier integrity is unknown. Here, we characterise the effects of distinct lactate isomers on HCAR1 trafficking and investigate its role on intestinal barrier function. In HEK293 cells expressing human HCAR1, both BRET and HTRF assays indicated L- and D-lactate differentially activate HCAR1-mediated Gai signalling. Confocal imaging and flow cytometry measurements demonstrated HCAR1 internalised from the plasma membrane in both a constitutive and ligand-independent manner, with L-lactate inducing 2.5 times greater internalisation than D-lactate. Neither internalisation (ligand-independent and dependent) nor ligand-induced signalling was altered in cells lacking β-arrestins. HCAR1 internalisation was dynamin-dependent, and its signalling was partially inhibited by the dynamin GTPase inhibitor, Dyngo-4a, implying HCAR1 may continue signalling following internalisation. Despite isomer-dependent differences in the levels of ligand-induced HCAR1 internalisation, both forms of lactate trafficked HCAR1 to a similar endosomal compartment. HCAR1 poorly co-localised with an early endosome marker, EEA1, and partially localised to APPL1 endosomes. The human intestinal epithelial barrier model cell line, Caco-2, expresses HCAR1 in both differentiated and undifferentiated cells, and preliminary data suggest HCAR1 is constitutively internalised. Sustained exposure to low concentrations of lactate increased transepithelial electrical resistance readings, suggesting improved barrier function. Translating the spatial regulation of HCAR1 into Caco-2 cells will enhance our knowledge of how HCAR1 responds to L- and D-lactate to influence local control of gut barrier integrity and inflammatory responses in the gut.