Endocrine Abstracts

Lactate is a metabolite that activates the G-protein coupled receptor, Hydroxycarboxylic acid Receptor 1 (HCAR1) to regulate physiological processes such as lipolysis, cancer cell survival, and neuroprotection. Lactate exists in two forms, L+ and D-, with the L isoform predominant in the human body. Interestingly, both isoforms are only found together in the gastrointestinal tract. L-lactate is synthesised as a by-product of anaerobic respiration, whereas D-lactate, is a product of fermentation by microbiota. To understand the different physiological roles of HCAR1 following activation by each form of lactate, our aim was to characterise the potential differential effects of L- and D-lactate on HCAR1 activity. HCAR1 mediates its signalling by coupling to Gαi to inhibit adenylate cyclase and reduce intracellular cAMP levels. Using HEK293 cells stably expressing FLAG-tagged HCAR1, we first investigated lactate-dependent differences in Gαi, Gαs and Gαq-protein activation. We confirmed HCAR1 activated only Gαi signalling, with L-lactate exhibiting a significantly higher potency than D-lactate (IC50 L-lactate: 23.67 mM, IC50 D-lactate: 61.84 mM, P<0.0001, T-test). In addition, two distinct HCAR1-selective ligands (HTL60092 and HTL61461) activated Gαi signalling with a higher potency than lactate (IC50 HTL60092: 245.9 nM, IC50 HTL61461: 18.6 nM, P<0.0001, T-test). Unexpectedly, measurement of intracellular calcium levels identified L- but not D-lactate could inhibit ATP-mediated calcium signalling without impacting ATP-mediated IP1 levels, suggesting possible crosstalk with a purinergic receptor downstream of Gαq/PLC activation. Analysis of HCAR1 trafficking via confocal microscopy indicates that HCAR1 undergoes both constitutive and lactate-induced internalisation, to an endosomal compartment that poorly co-localises with early endosomal markers. Preliminary data suggests that pre-treatment with ATP may promote D-Lactate-induced HCAR1 trafficking to the early endosome. Together, these results emphasise that L- and D-lactate activate HCAR1 in unique ways and both isomers must be considered to gain a better understanding of HCAR1 action in physiological settings.