3,5-DHBA

The lactate receptor HCAR1 modulates neuronal network activity through the activation of Galpha and Gbeta subunits.[Pubmed:30926749]

J Neurosci. 2019 Mar 29. pii: JNEUROSCI.2092-18.2019.

The discovery of a G protein-coupled receptor for lactate named hydroxycarboxylic acid receptor 1 (HCAR1) in neurons has pointed to additional non-metabolic effects of lactate for regulating neuronal network activity. In this study, we characterized the intracellular pathways engaged by HCAR1 activation, using mouse primary cortical neurons from wild-type (WT) and HCAR1 knock-out (KO) mice from both sexes. Using whole-cell patch-clamp, we found that activation of HCAR1 with 3-chloro-5-hydroxybenzoic acid (3Cl-HBA) decreased miniature excitatory postsynaptic current frequency, increased paired-pulse ratio, decreased firing frequency, and modulated membrane intrinsic properties. Using fast calcium imaging, we show that HCAR1 agonists, 3,5-dihydroxybenzoic acid (3,5-DHBA), 3Cl-HBA, and lactate decreased by 40% spontaneous calcium spiking activity of primary cortical neurons from WT but not from HCAR1 KO mice. Notably, in neurons lacking HCAR1 the basal activity was increased compared to WT. HCAR1 mediates its effect in neurons through a Gialpha-protein. We observed that the adenylyl cyclase-cAMP-protein kinase A axis is involved in HCAR1 down-modulation of neuronal activity. We found that HCAR1 interacts with adenosine A1, GABAB, and alpha2-adrenergic receptors, through a mechanism involving both its Gialpha and Gibeta subunits, resulting in a complex modulation of neuronal network activity. We conclude that HCAR1 activation in neurons causes a down-modulation of neuronal activity through presynaptic mechanisms and by reducing neuronal excitability. HCAR1 activation engages both Gialpha and Gibeta intracellular pathways to functionally interact with other Gi -coupled receptors for the fine tuning of neuronal activity.SIGNIFICANCE STATEMENTExpression of the lactate receptor HCAR1 was recently described in neurons. Here, we describe the physiological role of this G-coupled receptor (GPCR) and its activation in neurons, providing information on its expression and mechanism of action. We dissected out the intracellular pathway through which HCA1R activation tunes down neuronal network activity. For the first time, we provide evidence for the functional cross-talk of HCA1R with other GPCRs, such as GABAB, adenosine A1 and alpha2A adrenergic receptors. These results set HCAR1 as a new player for the regulation of neuronal network activity acting in concert with other established receptors. Thus, HCAR1 represents a novel therapeutic target for pathologies characterized by network hyperexcitability dysfunction, such as epilepsy.

Dual Properties of Lactate in Muller Cells: The Effect of GPR81 Activation.[Pubmed:30884529]

Invest Ophthalmol Vis Sci. 2019 Mar 1;60(4):999-1008.

Purpose: Besides being actively metabolized, lactate may also function as a signaling molecule by activation of the G-protein-coupled receptor 81 (GPR81). Thus, we aimed to characterize the metabolic effects of GPR81 activation in Muller cells. Method: Primary Muller cells from mice were treated with and without 10 mM L-lactate in the presence or absence of 6 mM glucose. The effects of lactate receptor GPR81 activation were evaluated by the addition of 5 mM 3,5-DHBA (3,5-dihydroxybenzoic acid), a GPR81 agonist. Western blot analyses were used to determine protein expression of GPR81. Cell survival was assessed through 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability assays. Lactate release was quantified by commercially available lactate kits. 13C-labeling studies via mass spectroscopy and Seahorse analyses were performed to evaluate metabolism of lactate and glucose, and mitochondrial function. Finally, Muller cell function was evaluated by measuring glutamate uptake. Results: The lactate receptor, GPR81, was upregulated during glucose deprivation. Treatment with a GPR81 agonist did not affect Muller cell survival. However, GPR81 activation diminished lactate release allowing lactate to be metabolized intracellularly. Furthermore, GPR81 activation increased metabolism of glucose and mitochondrial function. Finally, maximal glutamate uptake decreased in response to GPR81 activation during glucose deprivation. Conclusions: The present study revealed dual properties of lactate via functioning as an active metabolic energy substrate and a regulatory molecule by activation of the GPR81 receptor in primary Muller cells. Thus, combinational therapy of lactate and GPR81 agonists may be of future interest in maintaining Muller cell survival, ultimately leading to increased resistance toward retinal neurodegeneration.

Modulation of hippocampal excitability via the hydroxycarboxylic acid receptor 1.[Pubmed:29704292]

Hippocampus. 2018 Aug;28(8):557-567.

In addition to its prominent role as an energetic substrate in the brain, lactate is emerging as a signaling molecule capable of controlling neuronal excitability. The finding that the lactate-activated receptor (hydroxycarboxylic acid receptor 1; HCA1) is widely expressed in the brain opened up the possibility that lactate exerts modulation of neuronal activity via a transmembranal receptor-linked mechanism. Here, we show that lactate causes biphasic modulation of the intrinsic excitability of CA1 pyramidal cells. In the low millimolar range, lactate or the HCA1 agonist 3,5-DHBA reduced the input resistance and membrane time constant. In addition, activation of HCA1 significantly blocked the fast inactivating sodium current and increased the delay from inactivation to a conducting state of the sodium channel. As the observed actions occurred in the presence of 4-CIN, a blocker of the neuronal monocarboxylate transporter, the possibility that lactate acted via neuronal metabolism is unlikely. Consistently, modulation of the intrinsic excitability was abolished when CA1 pyramidal cells were dialyzed with pertussis toxin, indicating the dependency of a Galphai/o -protein-coupled receptor. The activation of HCA1 appears to serve as a restraining mechanism during enhanced network activity and may function as a negative feedback for the astrocytic production of lactate.

Lactate increases myotube diameter via activation of MEK/ERK pathway in C2C12 cells.[Pubmed:29377587]

Acta Physiol (Oxf). 2018 Jun;223(2):e13042.

AIM: Lactate is produced in and released from skeletal muscle cells. Lactate receptor, G-protein-coupled receptor 81 (GPR81), is expressed in skeletal muscle cells. However, a physiological role of extracellular lactate on skeletal muscle is not fully clarified. The purpose of this study was to investigate extracellular lactate-associated morphological changes and intracellular signals in C2C12 skeletal muscle cells. METHODS: Mouse myoblast C2C12 cells were differentiated for 5 days to form myotubes. Sodium lactate (lactate) or GPR81 agonist, 3,5-dihydroxybenzoic acid (3,5-DHBA), was administered to the differentiation medium. RESULTS: Lactate administration increased the diameter of C2C12 myotubes in a dose-dependent manner. Administration of 3,5-DHBA also increased myotube diameter. Not only lactate but also 3,5-DHBA upregulated the phosphorylation level of mitogen-activated protein kinase kinase 1/2 (MEK1/2), p42/44 extracellular signal-regulated kinase-1/2 (ERK1/2) and p90 ribosomal S6 kinase (p90RSK). MEK inhibitor U0126 depressed the phosphorylation of ERK-p90RSK and increase in myotube diameter induced by lactate. On the other hand, both lactate and 3,5-DHBA failed to induce significant responses in the phosphorylation level of Akt, mammalian target of rapamycin, p70 S6 kinase and protein degradation-related signals. CONCLUSION: These observations suggest that lactate-associated increase in the diameter of C2C12 myotubes is induced via activation of GRP81-mediated MEK/ERK pathway. Extracellular lactate might have a positive effect on skeletal muscle size.

A neuronal lactate uptake inhibitor slows recovery of extracellular ion concentration changes in the hippocampal CA3 region by affecting energy metabolism.[Pubmed:27559140]

J Neurophysiol. 2016 Nov 1;116(5):2420-2430.

Astrocyte-derived lactate supports pathologically enhanced neuronal metabolism, but its role under physiological conditions is still a matter of debate. Here, we determined the contribution of astrocytic neuronal lactate shuttle for maintenance of ion homeostasis and energy metabolism. We tested for the effects of alpha-cyano-4-hydroxycinnamic acid (4-CIN), which could interfere with energy metabolism by blocking monocarboxylate-transporter 2 (MCT2)-mediated neuronal lactate uptake, on evoked potentials, stimulus-induced changes in K(+), Na(+), Ca(2+), and oxygen concentrations as well as on changes in flavin adenine dinucleotide (FAD) autofluorescence in the hippocampal area CA3. MCT2 blockade by 4-CIN reduced synaptically evoked but not antidromic population spikes. This effect was dependent on the activation of KATP channels indicating reduced neuronal ATP synthesis. By contrast, lactate receptor activation by 3,5-dihydroxybenzoic acid (3,5-DHBA) resulted in increased antidromic and orthodromic population spikes suggesting that 4-CIN effects are not mediated by lactate accumulation and subsequent activation of lactate receptors. Recovery kinetics of all ion transients were prolonged and baseline K(+) concentration became elevated by blockade of lactate uptake. Lactate contributed to oxidative metabolism as both baseline respiration and stimulus-induced changes in Po2 were decreased, while FAD fluorescence increased likely due to a reduced conversion of FAD into FADH2 These data suggest that lactate shuttle contributes to regulation of ion homeostatsis and synaptic signaling even in the presence of ample glucose.

Hydroxybenzoic acids and their derivatives as peroxynitrite scavengers.[Pubmed:26461345]

Free Radic Biol Med. 2014 Oct;75 Suppl 1:S33-4.

A social challenge of the 21(st) century is to reduce the incidence of chronic diseases. A balanced diet rich in polyphenols could contribute to reduce the risk and to the prevention of diabetes, coronary heart disease, cancer, Alzheimer's diseases and cataract(1). Hydroxybenzoic acids (HBA) and their derivatives, which are one of the substances responsible for these beneficial properties, are known mainly due to their antioxidant properties(2). They are effective scavengers of free radicals and reactive nitrogen species, such as peroxynitrite. Peroxynitrite is resulting from the reaction of nitric oxide with superoxide, causes lipid peroxidation and subsequent cellular damage and is responsible for the inactivation of many enzymes, activation of stress signalling pathways, release of proapoptotic factors, as well as cardiovascular dysfunction in septic schock(3). In this study we have tested 2-HBA, 3-HBA, 4-HBA, acetylsalicylic acid, 4-HBA methyl and propyl esters, 2,3-dihydroxybenzoic acid (DHBA), 2,5-DHBA, 2,4-DHBA, 2,6-DHBA, 3,5-DHBA, 3,4-DHBA, gallic acid and caffeic acid, by UV/VIS spectroscopy. The best ability to scavenge peroxynitrite was observed for gallic acid, 2,4-DHBA, 3,5-DHBA and caffeic acid. Improved comprehension of the complex relationship between the antioxidant properties of substances and their structure is important to understand their proper use in the prevention and treatment of diseases and for the detection of pathological processes. Monitoring and improved understanding of the antioxidant properties of hydroxybenzoic acid derivatives are important due to their frequent use in modern medical nutrition therapies.

3,5-Dihydroxybenzoic acid, a specific agonist for hydroxycarboxylic acid 1, inhibits lipolysis in adipocytes.[Pubmed:22434674]

J Pharmacol Exp Ther. 2012 Jun;341(3):794-801.

Niacin raises high-density lipoprotein and lowers low-density lipoprotein through the activation of the beta-hydroxybutyrate receptor hydroxycarboxylic acid 2 (HCA2) (aka GPR109a) but with an unwanted side effect of cutaneous flushing caused by vascular dilation because of the stimulation of HCA2 receptors in Langerhans cells in skin. HCA1 (aka GPR81), predominantly expressed in adipocytes, was recently identified as a receptor for lactate. Activation of HCA1 in adipocytes by lactate results in the inhibition of lipolysis, suggesting that agonists for HCA1 may be useful for the treatment of dyslipidemia. Lactate is a metabolite of glucose, suggesting that HCA1 may also be involved in the regulation of glucose metabolism. The low potency of lactate to activate HCA1, coupled with its fast turnover rate in vivo, render it an inadequate tool for studying the biological role of lactate/HCA1 in vivo. In this article, we demonstrate the identification of 3-hydroxybenzoic acid (3-HBA) as an agonist for both HCA2 and HCA1, whereas 3,5-dihydroxybenzoic acid (3,5-DHBA) is a specific agonist for only HCA1 (EC(50) approximately 150 muM). 3,5-DHBA inhibits lipolysis in wild-type mouse adipocytes but not in HCA1-deficient adipocytes. Therefore, 3,5-DHBA is a useful tool for the in vivo study of HCA1 function and offers a base for further HCA1 agonist design. Because 3-HBA and 3,5-DHBA are polyphenolic acids found in many natural products, such as fruits, berries, and coffee, it is intriguing to speculate that other heretofore undiscovered natural substances may have therapeutic benefits.

3,5-Dihydroxybenzoic acid a potential biomarker for the consumption of many food products, including beer, nuts, peanut, and pulses.

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