GPR81 protein expression has previously been verified in isolated murine RGCs and Müller cells by immunostaining. Moreover, mRNA expression levels of GPR81 have also been quantified in retinas obtained from mice.
20 However, in this study, we aimed to verify the presence of the receptor and possible regulations in response to altered levels of energy availability. Deprivation of glucose led to increased GPR81 expression, indicating an amplification of lactate-mediated effects through GPR81 activation. The presence of extracellular lactate has previously been shown to increase Müller cell survival through uptake of lactate by MCT transporters,
13 thus indicating that lactate uptake is crucial for Müller cell viability. Moreover, studies on various tumor cell lines have shown that silencing GPR81 led to reduced survival,
19 which could support that lactate-mediated GPR81 activation is physiologically important in cell survival by activation of intracellular-mediated pathways. Thus, previous studies suggest that lactate uptake directly enhances cell survival, whereas lactate-mediated GPR81 activation may serve as a regulatory pathway that maintains the intracellular lactate homeostasis. To elucidate the role of GPR81 on primary mouse Müller cells, we investigated whether GPR81 activation would counteract the protective effects of lactate on cell survival. Surprisingly, the addition of 5 mM GPR81 agonist did not have any effect on survival, either cytotoxic or cytoprotective effects. To further investigate the link between GPR81 activation and lactate as a metabolite, lactate release and metabolism was explored. During physiological conditions, with sufficient amounts of glucose, lactate was released in high amounts corresponding nicely to the chosen lactate concentration of 10 mM in subsequent experiments with extracellular lactate exposure. During glucose deprivation, the extracellular concentration of lactate diminished significantly, thereby suggesting that lactate was metabolized internally instead of being released. Interestingly, GPR81 activation also resulted in decreased lactate release in the presence of glucose. In line with this, previous studies have shown that lactate-mediated activation of GPR81 on adipocytes inhibits lipolysis in vitro as well as in vivo,
34–36 and 3,5-DHBA has been verified as a specific agonist to facilitate this effect.
36 It has been proposed that GPR81 activation of adipocytes is to preserve energy as fat, especially during stress and short-term starvation.
37 During glucose deprivation in our study, we did not establish any significant effect on lactate release in response to GPR81 activation of the Müller cells. However, in the presence of physiological glucose levels, lactate release decreased significantly, thereby identifying that other metabolic pathways, besides fat metabolism, are affected in response to GPR81 activation. We furthermore elucidated the consequence of GPR81 activation on glucose metabolism and identified an increased glucose metabolism through oxidative phosphorylation in response to GRP81. Thus, GPR81 activation enhanced glucose metabolism through the TCA cycle while bypassing the production of lactate.