The G-protein-coupled lactate receptor, GPR81 (HCA1), is known to promote lipid storage in adipocytes by downregulating cAMP levels. Here, we show that GPR81 is also present in the mammalian brain, including regions of the cerebral neocortex and hippocampus, where it can be activated by physiological concentrations of lactate and by the specific GPR81 agonist 3,5-dihydroxybenzoate to reduce cAMP. Cerebral GPR81 is concentrated on the synaptic membranes of excitatory synapses, with a postsynaptic predominance. GPR81 is also enriched at the blood-brain-barrier: the GPR81 densities at endothelial cell membranes are about twice the GPR81 density at membranes of perivascular astrocytic processes, but about one-seventh of that on synaptic membranes. There is only a slight signal in perisynaptic processes of astrocytes. In synaptic spines, as well as in adipocytes, GPR81 immunoreactivity is located on subplasmalemmal vesicular organelles, suggesting trafficking of the protein to and from the plasma membrane. The results indicate roles of lactate in brain signaling, including a neuronal glucose and glycogen saving response to the supply of lactate. We propose that lactate, through activation of GPR81 receptors, can act as a volume transmitter that links neuronal activity, cerebral energy metabolism and energy substrate availability.
Lactate, acting as a buffer between glycolysis and oxidative metabolism, is exchanged as a fuel between cells and tissues, depending on glycolytic and oxidative rates (Brooks 2009). The brain exports lactate at rest, but once blood lactate levels rise, for example, during physical exertion, there is a net flux of lactate into the brain (Rasmussen et al. 2010; van Hall 2010). In the brain, astrocytes can produce lactate from glycogen stores for use by neurons and oligodendrocytes, which appear to require lactate in addition to glucose for optimal function, such as memory formation (Suzuki et al. 2011), and myelin production and sustenance of long axons (Lee et al. 2012; Rinholm et al. 2011). In addition, lactate is neuroprotective against various types of brain damage including ischemic (Schurr et al. 2001; Smith et al. 2003), excitotoxic, and mechanical insults (Ros et al. 2001; Cureton et al. 2010). Some of these effects cannot easily be interpreted in terms of the role of lactate as a fuel, but suggest that lactate has intercellular signaling roles. This notion is supported by the observation that the monocarboxylate transporter-2 (MCT2) is selectively co-located with glutamate receptors at the postsynaptic membranes of fast acting excitatory synapses (Bergersen et al. 2001, 2005). Further, lactate is known to mediate cerebral vasodilatation causing increased brain blood flow (Gordon et al. 2008). The notion of multiple signaling roles of lactate in brain leads to the concept of lactate being a “volume transmitter” of metabolic information (Bergersen and Gjedde 2012). Lactate signaling may occur through several mechanisms, including modulation of prostaglandin action (Gordon et al. 2008), redox regulation (Brooks 2009), and activation of the lactate responsive G-protein-coupled receptor GPR81.
The lactate selectivity of the orphan receptor GPR81, also known as hydroxycarboxylic acid receptor 1 (HCA1, IUPHAR nomenclature), was discovered in adipose tissue where GPR81 is highly expressed and serves to downregulate the formation of cAMP, thereby curbing lipolysis and promoting storage of energy-rich metabolites (Ahmed et al. 2009, 2010). GPR81 mRNA is expressed at lower levels in other tissues, including brain: evidence from in situ hybridization suggests a wide distribution of GPR81 mRNA in the brain, including in the principal neurons in the cerebral cortex, hippocampus, and cerebellum (The Allen Institute for Brain Science, http://www.brain-map.org; GENSTAT, http://www.ncbi.nlm.nih.gov/gensat; St. Jude Children's Research Hospital, http://www.stjudebgem.org). However, the localization and function of the receptor in the brain has not been reported, nor its ultrastructural localization in brain or adipose tissue.
Here, we show for the first time the subcellular localization and the function of the lactate receptor GPR81 protein in brain cells. l-Lactate caused a dose-dependent reduction of cAMP in hippocampal slices, which is consistent with activation of the Gi-coupled receptor GPR81. The newly identified selective GPR81 agonist, 3,5-dihydroxybenzoic acid (3,5-DHBA), showed a concentration-effect curve similar to that previously observed in GPR81 expressing cells (Liu et al. 2012). In brain cells and in adipocytes, GPR81 immunoreactivity is concentrated at the plasma membrane as well as over intracellular vesicular organelles, suggesting the presence of reservoirs for trafficking of the receptor to and from the cell surface. The density of GPR81, represented by immunogold particles, at luminal and at abluminal membranes of cerebrovascular endothelial cells was about twice the density at membranes of astrocytic vascular end-feet. The highest GPR81 densities in brain were observed at excitatory synapses in hippocampus and cerebellum, about 7 times the densities at vascular endothelial membranes, with predominance at the postsynaptic membrane. The findings indicate a GPR81-mediated action of lactate, linking synaptic function, energy metabolism, and cerebral blood flow. Part of the results have been presented in abstract form (Bergersen et al. 2012).
Materials and Methods
All experimental procedures were approved by the section for comparative medicine at the Oslo University Hospital and the Norwegian animal research authority, conducted by FELACA C-certified operators, and complied with national laws and institutional regulations governing the use of animals in research.
An anti-mouse GPR81 antibody produced in rabbit, GPR81-S296 (Sigma-Aldrich), was used. This antibody recognizes the C-terminal part of the protein (amino acids 276–329). Two other commercial antibodies to GPR81 were tested, but found to give unsatisfactory results on western blotting.
Wild-type C57Bl/6 mice (6–8 weeks) were deeply anesthetized with pentobarbital (2 mL/kg, i.p.) and transcardially perfused with 4% paraformaldehyde in sodium phosphate buffer (NaPi, 0.1 M, pH 7.4) for 10 min. The brains were gently removed and stored in the fixative at 4 °C until use. Prior to sectioning, the brains were rinsed in NaPi and cryoprotected by sequential immersion in a 10% (2 h), 20% (3 h), and 30% (overnight) sucrose in NaPi solution. Sagittal brain sections were cut at 40 µm thickness on a freezing microtome (Microm, USA) and subjected to immunohistochemistry by the avidin–biotin peroxidase method (Hsu and Raine 1981) with modifications. Briefly, free floating sections were treated with a 3% hydrogen peroxide solution to inactivate intrinsic peroxidase activity; then treated with 1 M ethanolamine to neutralize free aldehyde groups. Unspecific binding sites were blocked by incubating the sections in PBS containing 3% normal goat serum, 1% BSA, 0.5% Triton X100 for 1 h. The sections were then incubated with the GPR81 antibody S296 (Sigma-Aldrich, Germany) (diluted 1:500 in 3% normal goat serum, 1% BSA, 0.1% Triton X100) overnight and then with an anti-rabbit biotinylated secondary antibody for 1 h (RNP1004V, diluted 1:100, GE Healthcare, UK) before they were treated with a streptavidin-biotin horseradish peroxidase complex (RPN 1051V, diluted 1:100, GE Healthcare, UK). The chromogen used was 3,3′-diaminobenzidine (Sigma-Aldrich, Germany). In the negative control, the primary antibody was omitted, all other steps where otherwise identical. The sections were mounted on gelatine-coated glass slides and scanned with a Mirax slide scanner (Zeiss, Germany) to obtain high-resolution images. Images G and H in Figure 2 are taken with an Axiocam camera (Zeiss), ensuring the same light intensity and exposure time for both images.
Quantitative Real-Time PCR
Total RNA for real-time RT-PCR was isolated from freshly dissected hippocampus, cerebellum, cerebral cortex (samples contained most of the neocortex), or abdominal fat from C57 BL/6 mice (n = 3), using RNeasy® Lipid Tissue Mini Kit (Qiagen) in combination with RNase-Free DNase Set (Qiagen) in accordance with the manufactures recommendations. RNA-quality was checked on 1% agarose gel. cDNA was produced from the isolated RNA using a High Capacity RNA-to-cDNA Kit (Applied Biosystems). Target-gene was identified and amplified using predesigned probes and primers from TaqMan-mix for GPR81 (Mm00558586_s1) and GAPDH (Mm99999915_g1) which was used as an endogenous control for normalization (both Applied Biosystems). RT-PCR reactions were carried out in a 20-μL mixture containing TaqMan® Gene Expression Master Mix (Applied Biosystems), 1 µL TaqMan-mix with 50 ng cDNA, RNA or nuclease free water. All reactions were done in triplicates, with samples from 3 different animals in each group. Negative controls with water were performed for each target. Controls with RNA-template were used to verify that the samples did not contain genomic DNA contamination. If the Ct-value (Cycle threshold-value) was <10 between cDNA and RNA sample, the data and sample were discarded. Standard curves with a 5-point 1:10 dilution series, starting at 100 ng were performed for each target. Default PCR program settings were used. All reactions were run on a StepOne Plus Real-Time PCR system (Applied Biosystems) using the default settings recommended by the manufacturer, and analyzed using StepOne software v2.2.2. Data were calculated based on the standard curves (standard-curve method), and target of interest was normalized against a controltarget gene (GAPDH). Standard curves with R2-values <0.99 were rejected.
C57 BL/6 adult mice were deeply anesthetized and decapitated. The brains were quickly removed, frozen in liquid nitrogen and stored at −80 °C until use. Omental adipose tissue was harvested from the same mice. Frozen brains and adipose tissue were homogenized in 1% SDS in NaPi solution containing a cocktail of protease inhibitors (Complete™, Roche, Germany) using a Dounce homogenizer. The homogenates were then sonicated and subjected to total protein quantification using the BCA protein assay (Pierce, Thermo Fisher Scientific, USA). Proteins in the adipose tissue homogenate were extracted with chloroform/methanol/water according to Wessel and Flügge (1984) prior to protein quantification. As an additional test of the specificity of the antibodies, we overexpressed GPR81 in HeLa cells, used using a murine GPR81 plasmid (Jeninga et al. 2009). HeLa cells were transfected using X-tremeGENE 9 transfection reagent (Roche) in accordance with the manufacturer's recommendations. Briefly, cells were seeded out in 10-cm dishes for an appropriate density, and 5 µg mGPR81 plasmid were used together with a 6:1 ratio transfection reagent: serum-free medium. Cells were harvested after 3 days, washed twice in cold PBS, pelleted, and snap-frozen until further use. Expression of GPR81 in transfected cells was validated using qPCR, as described above, with 2 exceptions: RNA was isolated from the cells using RNeasy Mini Kit (Qiagen) and TaqMan-mix for human GAPDH (Cat.-No.:4333764, Applied Biosystems) was used as controltarget. Both transfected and untransfected HeLa cells (∼1 million cells) were lysed with 1 mL cold RIPA buffer (25 mM Tris HCl pH7.6, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% Na deoxycholate and protease inhibitor cocktail) for 15 min and centrifuged at 14 000 × g for 15 min to pellet cell debris. The supernatant was transferred to new eppendorf tubes and subjected to total protein quantification using the BCA protein assay. The homogenates (10 and 20 µg total protein) were subjected to SDS PAGE electrophoresis (12.5% Criterion gel) and western blotting using the Criterion gel and blotting system (Bio-Rad Laboratories, USA). After incubation of the nitrocellulose membranes with the GPR81 antibody and secondary antibody (GE Healthcare anti rabbit IgG from donkey, HRP linked, NAV934V, 1:20000), the immunoreactive bands were detected using the chemiluminescence kit ECL SuperSignal (Thermo Scientific, USA) with a Fujifilm LAS3000 imager (Fujifilm, Japan).
Whole brains from C57Bl/6 mice were immersion fixed in 10% (v/v) neutral buffered formalin (Richard-Allan Scientific) for at least 48 h, and paraffin embedded. Brains were cut into 4-µm-thick sections using a rotary microtome (Microm 355 S), and sections were dried onto glass slides at 37 °C overnight. Sections were deparaffinized by heating at 57 °C for 10 min and incubated twice in Clear Rite 3-solution (Richard-Allan Scientific) for 5 min each. Thereafter, sections were rehydrated by incubating for 1 min each in a series of descending concentrations of ethanol (100%, 96%, and 70%), ending in distilled H2O (dH2O). Sections were then subjected to antigen retrieval by heating for 15 min at 95 °C in a Tris–EDTA buffer (10 mM Tris base, 3.8 mM EDTA, pH adjusted to 9.0), cooled at room temperature for 10 min, and incubated for 5 min in dH2O and 5 min in PBS/0.1% Tween-20. Sections were then treated with 0.1% Triton X-100 (Sigma) in PBS for 15 min and washed twice with 0.1% Tween 20 in PBS. Unspecific labeling was blocked by incubating in a solution containing 5% bovine serum albumin (BSA, Sigma), 5% goat serum (Sigma) and 0.1% Triton X-100 (Sigma). Sections were incubated overnight at 4 °C with primary antibodies diluted in 0.5% BSA/0.5% goat serum/0.1% Tween-20 in PBS. After washing, sections were incubated with Alexa Fluor 594-conjugated goat anti-mouse IgG (H + L) (Invitrogen, A11005) or Alexa Fluor 488-conjugated goat anti-rabbit IgG (H + L) (Invitrogen, A11008) secondary antibodies diluted 1:500, as appropriate. Cell nuclei were visualized by incubating with 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI, Sigma). Sections were coverslip-mounted using Mowiol 4–88 Reagent (Merck Biosciences Ltd) and examined with a Zeiss Axiovert 200 M microscope. Images were acquired and analyzed using AxioCamMRm and AxioVision Rel. 4.6 software. All microscope and software settings were equal for images being compared.
Antibodies against the following proteins were used at the indicated concentrations: GPR81 (GPR81-S-296, Sigma, SAB1300090, dilution 1:100), microtubule-associated protein 2 (MAP2; Sigma, M4403, dilution 1:1000) and glutamine synthetase (GS; BD Bioscience, ab610518; dilution 1:1000).
Electronmicroscopic Postembedding Immunogold Cytochemistry
Adult (4 months) C57 BL/6 mice (30 g, n = 3) were used. Animals were deeply anesthetized by an intraperitoneal injection of pentobarbital (0.4 mL/100 g body wt) and subjected to transcardial perfusion with a mixture of 0.1% glutaraldehyde and 4% formaldehyde (freshly depolymerized from paraformaldehyde) in 0.1M NaPi (pH 7.4) at 4 °C. The brains were left in situ overnight (4 °C).
Small rectangular pieces (typically 0.5 × 0. 5 × 1 mm) of brain tissue were cryoprotected by immersion in graded concentrations of glycerol (10%, 20%, and 30%) in 0.1M NaPi at 4 °C. Samples were then plunged into liquid propane cooled at −190 °C with liquid nitrogen in a Universal Cryofixation System KF80 (Reichert-Jung, Wien, Austria). Tissue blocs were moved with precooled forceps. For freeze-substitution (Bergersen et al. 2008), tissue samples were immersed in a solution of anhydrous methanol and 0.5% uranyl acetate overnight at −90 to −45. The subsequent steps were performed at −90 to −45 °C. Tissue samples were washed several times with anhydrous methanol to remove residual water and uranyl acetate. The infiltration in Lowicryl HM20 went stepwise from Lowicryl:methanol 1:2, 1:1, and 2:1 (1 h each) to pure Lowicryl (overnight). For polymerization, the tissue was placed in a precooled embedding mall. The polymerization was catalyzed by UV light at a wavelength of 360 nm for 2 days at −45 °C followed by 1 day at room temperature. Ultrathin sections (70 nm) were cut with a diamond knife on a Reichert–Jung ultramicrotome and mounted on nickel grids using an adhesive pen (David Sangyo).
Grids with the ultrathin sections were processed at room temperature in solutions containing 50 mM Tris HCl buffer, pH 7.4, 0.05M NaCl, and 0.1% Triton X-100 (TBST) and completed as stated below. Sections were first washed in TBST containing 0.1% sodium borohydride and 50 mM glycine for 10 min. They were then incubated overnight with primary antibodies diluted in TBST containing 2% HSA. Rabbit antibodies against GPR81 (diluted 1 µg/mL) were used. Goat anti-rabbit immunoglobulins coupled to 10-nm gold particles (BBI) were diluted 1:20 in TBST containing 2% HSA and 5 mg/mL ployethyleneglycol. In double-labeling experiments, sections were treated with the antibodies against GPR81 and mouse antibody against GS (1:1000). Both goat anti-rabbit immunoglobulins coupled to 10-nm gold particles and goat anti-mouse immunoglobulins coupled to 20-nm gold particles (BBI, Cardiff, UK) were diluted 1:20 in TBST containing 2% HSA and 5 mg/mL polyethyleneglycol.
Ultrathin sections were contrasted in uranyl acetate (15min) and lead citrate (90s), before they were observed in a FEI Tecnai 12 electron microscope. Electron micrographs were taken at a primary magnification of ×26 500.
Quantitative analysis: Electron micrographs were taken randomly in the stratum radiatum of hippocampus CA1, and in the molecular layer of the cerebellum in sections from 3 mice. Gold particles representing GPR81 immunoreactivity were quantified in the image analysis program ImageJ (National Institutes of Health, Bethesda, MD) as number of gold particles/µm of transacted membrane in luminal and abluminal membranes of micro vessels and in the surface membrane of perivascular end-feet of astrocytes, as well as in hippocampal asymmetric synapses representing Schaffer collateral/commissural system synapses and in parallel fiber–Purkinje cell synapses of the cerebellum (for identification criteria of parallel fiber–Purkinje cell synapses, see Bergersen et al. 2001). A plugin for ImageJ (http://rsb.info.nih.gov/ij/) was used to outline the plasma membrane of nerve terminals and PSDs. Recorded coordinates were submitted to a program written in Python (http://www.python.org) for computation of particle densities (number of gold particles per µm2) (Larsson and Broman 2005). The source code of the ImageJ plugin and the Python program is available at http://www.neuro.ki.se/broman/maxl/software.html. Specific membrane compartments were defined for quantification (see schematic illustration in Fig. 1). They correspond to the postsynaptic membrane, defined as overlying the postsynaptic density (PSD), the facing presynaptic membrane, and perisynaptic membranes (corresponding to the plasma membrane on each side of the PSD). Perisynaptic membranes were defined for convenience as half the width of the PSD for all synapses. Particles situated with their centers within 30 nm of the midline of the membrane were recorded, corresponding to the resolution of the immunogold method. At the capillaries, only particles on the cytoplasmic side of the plasma membrane were counted. The “background” immunogold particle density was recorded in the same way along 500-nm long lines randomly placed by the program drawn across the observed electron micrographs (n = 20), avoiding cell membranes and mitochondria estimating the average labeling of the tissue, rather than unspecific labeling. Only synaptic profiles with clearly visible postsynaptic membrane and PSD were selected for quantitative analysis (the 20 first encountered in each of the 3 animals and regions). All of the microvessels (7–9 in each animal and region) were recorded. To estimate the distribution of GRP81 in the proximo-distal dimension, the distance between the centers of gold particles representing GPR81 and the external face of the postsynaptic membrane was measured along an axis perpendicular to the PSD and sorted into 10 nm bins. All gold particles located within the postsynaptic dendritic spines and the presynaptic terminals were recorded.
Statistical significance was determined by ANOVA analysis with GraphPad software (http://www.graphpad.com/demos/).
cAMP Analysis After GPR81 Activation in Brain Slices
Experiments with hippocampal slices were performed as previously described (Gundersen et al. 1991; Morland et al. 2013). For each experiment, one male Wistar rat, 6 weeks of age, (Scanbur, Sollentuna, Sweden) was anesthetized with isofluran (Florene; AbbVie Ltd, UK) and decapitated. The brains were gently removed, and the hippocampi were dissected out on ice and cut into 300-µm thick slices with a Sorvall tissue chopper (Sorvall, Newtown, CT, USA). The slices were preincubated in oxygenated Krebs' solution (126 mM NaCl, 3 mM KCl, 10 mM NaPi, 1.2 mM CaCl2, and 1.2 mM MgSO4 and 10 mM glucose) for 30min at 30 °C. Subsequently, assay of cAMP generation was initiated, and the slices were incubated for 16min at 30 °C in the same buffer (basal) or in buffer supplemented with forskolin (10 µM) alone, or with forskolin (10 µM) and increasing concentrations of l-lactate (0.1–30 mM), or increasing concentrations of 3,5-DHBA (0.01–10 mM). To block degradation of cAMP generated during the experiment, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM) was included in all preincubation and incubation steps.
The assay was stopped by transferring the tissue sections to vials containing ice cold HCl (0.1 M; 150 µL). The sections were subsequently homogenized, sonicated in an ultrasonic water bath (10 min), and centrifuged (17 500 × g; 10 min). The supernatants were transferred to new tubes, and protein concentrations were determined by the Micro BCA Protein Assay Kit (Thermo Scientific). Each sample (100 µL) was evaporated to dryness in a SpeedVac system (Termo Savant) and subsequently dissolved in assay buffer (sodium acetate, pH 6.2; provided with the cAMP FlashPlate kit; PerkinElmer, Inc., MA, USA). The cAMP contents of the samples were determined by radioimmunoassay using the cAMP FlashPlate scintillation proximity system (PerkinElmer, Inc., MA, USA). Each sample was assayed in duplicate. Curve-fit analysis (nonlinear regression/4-parameter logistic) was performed with the GraphPad Prizm 5.0 software (http://www.graphpad.com/demos/).
Regional and Cellular Distribution
We examined the cellular and regional distribution pattern of GPR81 in sagittal sections of adult male WT C57/BL6 mice (Figs 1 and 2A). GPR81 staining was observed throughout the brain with some regions showing more prominent labeling. Strong labeling was present in the cerebellar Purkinje neurons and their dendrites (Fig. 2B), the pyramidal cells in the hippocampus (Fig. 2C), in neurons in the dentate hilus (Fig. 2D), and in the cerebral neocortex (Fig. 2E,G). In contrast, neurons in the striatum were barely distinguishable from the general labeling of the neuropil (Fig. 2F). In addition, the GPR81 antibody produced strong labeling of neurons in some brain stem regions, such as the substantia nigra (not shown).
Expression of GPR81 mRNA and Protein in Brain and Adipose Tissue
GPR81 mRNA as well as protein was found in all the regions of the brain that were investigated. GPR81 mRNA was expressed to a similar degree in both cerebellum and hippocampus, but about 60% lower in the cerebral neocortex than in cerebellum and hippocampus (Fig. 3A), in agreement with the results from the GPR81-immunolabeling shown in Figure 2. Omental white adipose tissue was used as a positive control and showed ∼140 fold of the mRNA level in cerebellum (Fig. 3B), relative to the total mRNA extracted. These results verify published data showing that GPR81 is highly expressed in adipose tissue compared with whole brain (Liu et al. 2009), but also reveal that there are variations between different parts of the brain regarding GPR81 expression.
GPR81 protein was demonstrated by western blotting (Fig. 3C). Three anti-GPR81 antibodies were tested but only one of them gave a band at the appropriate molecular mass (about 39 kDa) and was therefore used for immunotechniques in this study. The specificity of the anti-GPR81 antibody was tested by western blotting of mouse brain and adipose tissue as well as of HeLa cells that overexpressed the murine GPR81 protein. In the brain, the strongest band was at a molecular mass of about 39 kDa, which corresponds to the predicted molecular mass of GPR81. This band is also strong in the adipose tissue sample and in HeLa cells transfected with GPR81. A higher molecular mass band (∼60 kDa) is present in brain and adipose tissue. This band is also strong in the transfected HeLa cells but weak in native HeLa cells and is therefore probably a form of GPR81, possibly a glycosylated form of the receptor. To our knowledge, the glycosylation level of GPR81 has not yet been described in detail; however, it is well known that G protein-coupled receptors (GPCRs) are glycosylated (Wheatley and Hawtin 1999), increasing the molecular mass of the protein on SDS-PAGE by amounts consistent with the ∼60 kDa band observed here (Tansky et al. 2007). The lower molecular mass band seen in adipose tissue as well as the very small fragments at the bottom of the gel seen in transfected cells and brain tissue could represent fragments of the protein. In the literature, most western blot analyses of GPR81 have been performed using liver extract or cell lysate, and to our knowledge, this is the first western blot demonstration of GPR81 in brain and adipose tissue extracts.
GPR81 is Present in Neurons, Astrocytes and Capillaries
To determine the cellular localization of GPR81, we performed double-labeling immunofluorescence microscopy of cerebellar and hippocampal cortex, the regions that showed the highest overall expression of GPR81 mRNA. Immunostaining of paraffin-embedded sections demonstrated the presence of GPR81 in neurons in both hippocampus and cerebellum. There was extensive co-localization with the neuronal marker MAP2, indicating the presence of GPR81 in neurons. The GPR81-like immunoreactivity was particularly strong on cell bodies and proximal parts of dendrites of hippocampal pyramidal cells and cerebellar Purkinje cells (Fig. 4A,C). Double-labeling for GPR81 with the astrocyte marker GS, revealed the presence of GPR81 also in astrocytes and at capillaries (Fig. 4B,D).
Electronmicroscopic Localization of GPR81 in Brain and Adipose Tissue
Adipocytes from rat omental fat, included as a positive control, showed GPR81 concentration along the surface membranes as well as in subplasmalemmal vesicular organelles (Fig. 5A). Little or no labeling occurred over lipid droplet areas or extracellular connective tissue space with collagen fibrils. In the brain, GPR81 was concentrated along the plasma membranes of vascular endothelial cells, the luminal as well as the abluminal leaflets, and on the surface membranes of perivascular astrocytic end-feet (Fig. 5B). The density was about twice as high in endothelial cell membranes, compared with membranes of astrocytic end-feet (Fig. 5C,D). There was no significant difference between hippocampus and cerebellum. In the nervous tissue, GPR81 was highly concentrated at synaptic membranes of excitatory-type synapses, in hippocampus as well as in cerebellum (Fig. 5G,H). The pre- and postsynaptic membranes had much higher immunogold particle densities than the immediately adjacent perisynaptic membranes, both in hippocampus and cerebellum. In the proximo-distal direction, perpendicular to the postsynaptic membrane, the highest particle density was at the postsynaptic membrane, with high density also in the spine and an additional peak in the nerve ending (Fig. 5I).
To further examine the astrocytic labeling suggested by immunofluoresence microscopy, we performed electron microscopic double-labeling for GPR81 and GS (Fig. 6A,B). Perisynaptic processes of astrocytes, identified by GS immunoreactivity, showed GPR81 signaling gold particles along their plasma membranes. Owing to the limited resolution of the immunogold method, some gold particles may possibly represent immunoreactivity in adjacent structures. Nevertheless, the observation indicates a low GPR81 density not only in the perivascular, but also in the perisynaptic processes of astrocytes.
Hippocampal Cortex Contains Functional GPR81 Receptors
As we found expression of GPR81 to be highly concentrated in hippocampal cortex, we investigated whether the endogenous agonist, l-lactate, as well as the selective agonist, 3,5-DHBA (Liu et al. 2012), could inhibit cAMP synthesis in acute hippocampal slices in vitro. Rat hippocampal slices were exposed to forskolin (10 µM), an activator of adenylyl cyclase (cAC), and the absence or presence of increasing concentrations of l-lactate (0.1–30 mM) or 3,5-DHBA (0.01–10 mM). Samples incubated in the absence of both forskolin and agonist were also included record basal cAMP synthesis. Both agonists caused concentration-dependent inhibition of forskolin-stimulated cAMP generation (Fig. 7). 3,5-DHBA, the more potent agonist, inhibited forskolin-stimulated cAMP generation with half-maximal inhibition (IC50) at 1.4 mM (95% confidence interval 0.58–3.3 mM). Analysis of the data points revealed a sigmoidal curve fit (4-parameter logistic) consistent with a single-site receptor interaction (R2 ≈ 0.94; Hill slope = −0.8 ± 0.2, mean ± SEM). l-Lactate displayed substantially lower potency than 3,5-DHBA. The maximal inhibitory effect of l-lactate was therefore not determined, since the highest concentrations of l-lactate employed in the assay (30 mM) were not high enough to cause full inhibition of cAMP generation. Experiments with higher concentrations of l-lactate were not performed as concentrations above 30 mM may exert actions not related to orthosteric receptor activation (Liu et al. 2012). Importantly, concentrations of l-lactate within the physiological range elicited robust inhibition of forskolin-stimulated cAMP generation in the hippocampal slices. The differences in potency of 3,5-DHBA and l-lactate are consistent with previous characterization of GPR81 in transfected SK-N-MC cells stably expressing GPR81 (Liu et al. 2012).
We show, for the first time, that the G-protein-coupled lactate receptor GPR81 is present and active, not only in adipocytes, but also in brain cells. We further show, also for the first time, that GPR81 is located on the plasma membrane as well as on intracellular vesicular organelles implying trafficking of the receptor between intracellular stores and the surface membrane. In the brain parenchyma, the highest concentration of GPR81 is at the synaptic membrane and in intracellular vesicular organelles of excitatory synapses in hippocampal and cerebellar cortex. These regions show the highest levels of GPR81 mRNA as well as of GPR81 protein in the brain. We provide evidence that l-lactate, at physiologically relevant concentrations can activate the GPR81 receptor, causing reduction of cAMP levels, thus proving that GPR81 is functional in the brain at high enough levels to mediate physiological effects. Similarly, the GPR81 selective agonist, 3,5-DHBA, caused a sigmoidal dose–response curve, with Hill slope near 1, consistent with the single-site receptor interaction.
Co-localization with markers of neurons (MAP2) and glia (GS) indicate a predominant localization of GPR81 in neuronal structures and a lesser localization in astrocytes. At the blood–brain barrier, GPR81 density is similar at the luminal and abluminal parts of the endothelial plasma membrane, and lower at the membranes of vascular end-feet of astrocytes.
The role of lactate in brain energy metabolism is controversial (Gjedde and Marrett 2001; Gjedde et al. 2002; Hertz 2004; Jolivet et al. 2010; Mangia et al. 2011). An astrocyte-neuron-lactate shuttle, implying that lactate derived from astrocytes can enter neurons where it serves as a substrate of oxidative metabolism, was introduced by Pellerin and Magistretti (1994), suggesting that lactate is an astrocyte-derived fuel for neurons under normal circumstances (Bergersen 2007; Brown and Ransom 2007; Pellerin et al. 2007; Choi et al. 2012). However, this claim may not take full account of the fact that glucose also enters neurons (Simpson et al. 2007), where it is metabolized through the glycolytic pathway, leading to the production of lactate in neurons (Gjedde and Marrett 2001), with uncertain contribution from extraneuronal lactate. Conversely, pyruvate can be fully oxidized in astrocytes (Hertz et al. 2007), questioning the exclusive role proposed for astrocytes as source of intraneuronal pyruvate.
We suggest that lactate, in addition to its role as a fuel for neurons, is a “volume transmitter” of metabolic states in the brain. In contrast to conventional “wired” neurotransmitters, volume transmitters are thought to act outside classical synapses, and to exert neuromodulatory effects on larger population of neurons. A cue to the nature of this role is the observation that lactate regulates cerebral blood flow (Gordon et al. 2008), serving as a signal rather than as a metabolic fuel. The present finding of GPR81 receptors in widespread regions of the brain, especially in the hippocampal cortex and cerebellum, and in different cellular compartments stimulates new thinking about lactate function. Most work on GPR81 has focused on adipose tissue, in which lactate is known to inhibit lipolysis through GPR81-mediated lowering of the cAMP level (Cai et al. 2008; Ahmed et al. 2009; Liu et al. 2009).
In the current study, we demonstrate that GPR81 immunoreactivity in the brain represents functional GPR81 receptors, mediating inhibition of cAC. The relative potencies of l-lactate and 3,5-DHBA as inhibitors of forskolin-stimulated cAMP generation are congruent with the known characteristics of the GPR81/HCA1 receptor (Liu et al. 2012). Furthermore, 3,5-DHBA is an agonist specific for GPR81/HCA1, that is, it does not display efficacy at other hydroxycarboxylic acid receptors (HCA2/GPR109a and HCA3/GPR109b) (Liu et al. 2012). The IC50 observed for 3,5-DHBA and predicted for l-lactate were somewhat higher in the brain slices than those previously reported from stably transfected cells expressing recombinant human GPR81/HCA1 (Liu et al. 2012). These differences may relate to variations associated with species difference, possibly altered glycosylation of recombinantly expressed receptors, as well as with the experimental systems, that is, brain slices, a multicellular preparation presenting a relative diffusion barrier, compared with a monolayer cell culture.
Altogether, the current study provides compelling evidence that activation of cerebral GPR81 receptors inhibits cAMP generation, hence slowing glycolysis rates when lactate concentrations rise. Many of the observed effects of lactate on neuronal function could result from receptor-mediated responses, either through GPR81 or through other yet unidentified receptors. The observation that lactate administration provides protection in ischemia (Schurr et al. 1997, 2001; Cureton et al. 2010) has been ascribed to metabolic roles of lactate, such as enhanced neuronal energy production. However, this effect is not readily explained by increased metabolism, as lactate use is oxygen-dependent, and thus inhibited under ischemic conditions. Similarly, the observed protective effect of lactate on glutamate toxicity in the brain (Ros et al. 2001) could be partly due to receptor-mediated mechanisms, and not only to the ability of lactate to meet an increased energy demand of these neurons that are overactivated by exposure to high concentrations of glutamate (Schurr et al. 1999). During microdialysis of the extracellular space of rat cerebral cortex, excitotoxic concentrations of glutamate raised the concentration of lactate and lowered the concentration of glucose in the dialysate (Ros et al. 2001). The size of the lesion was reduced and the decrease in glucose prevented by adding l-lactate. This glucose sparing effect of l-lactate may, in part, have been mediated through downregulation of cAMP by GPR81. Further, physiological stimulation of climbing fibers causes glutamate receptor-dependent lactate production from glucose in the cerebellar cortex (Caesar et al. 2008), suggesting that a rise in extracellular lactate in response to neuronal activation is a general phenomenon. Extracellular lactate may stimulate cerebral vasodilatation (Gordon et al. 2008), providing a possible link between neuronal activation and rise in cerebral blood flow.
Likewise, the suppression of noradrenalin and adrenalin release by “clamping” blood lactate at 4 mM (Fattor et al. 2005), suggests the presence of a lactate receptor-dependent mechanism. The ability of lactate to reduce cAMP levels through GPR81 activation could underlie this effect. Interestingly, β-adrenoceptor blockers, which inhibit catecholamine-stimulated cAMP synthesis, are neuroprotective in stroke and other brain injuries, and also cause decreased extracellular glutamate levels, an effect that may further contribute to limit excitotoxic cell damage (Goyagi et al. 2011). These effects may be mimicked by the cAMP reduction caused by lactate through GPR81.
The well-known monoaminergic volume transmitter dopamine is released at extrasynaptic and some synaptic sites and diffuses considerable distances to reach its receptors (Fuxe et al. 2010). Our observations on the lactate receptor GPR81 suggest that lactate acts in a similar manner: it is released from neurons or astrocytes via MCT-mediated transport under conditions where glycolysis prevails. In the extracellular space, lactate diffuses and binds to the GPR81 lactate receptors at different distances from the sites of release. Both the degradation of lactate-by-lactate dehydrogenase and the facilitated diffusion by MCTs mediate near-equilibrium processes, which dissipate lactate concentration differences across cell membranes and tissue volumes. These processes are further enhanced by the connection of astrocytes into a functional syncytium through gap junctions. In this respect, lactate behaves somewhat like nitric oxide, which is also a readily metabolized volume transmitter that rapidly diffuses across cell membranes to distant cells, where it activates its effector molecule, guanylyl cyclase (Garthwaite 2008).
In adipose tissue, which has the highest GPR81 density in the body, and where most of the extant evidence on the lactate receptor GPR81 originated, the receptor appears to serve the distinct function of promoting energy storage as fat: during postprandial insulin-induced glucose uptake and subsequent glycolysis, adipocytes release lactate as an autocrine mediator acting on GPR81 to lower cAMP and thereby cause reduced lipolysis (and glycolysis). In the brain, one function of lactate volume transmission may be to serve as a metabolic sensor engaged in overflow control. Through its action on GPR81, lactate can curb glycolysis when it runs too fast compared with oxidative phosphorylation, and thereby prevent a further increase in the level of lactate and the concomitant acidification. This function may be particularly important in the brain where low pH perturbs synaptic transmission, including interference with glutamate receptor function (Traynelis and Cull-Candy 1991).
A receptor complementary to GPR81 is the bicarbonate-responsive soluble AC, recently proposed to stimulate glycolysis in astrocytes in response to neuronal activity, yielding increased availability of lactate to neurons. Neuronal activity raises extracellular bicarbonate and K+ levels, which in turn activate astrocytic bicarbonate uptake (Choi et al. 2012) and thus soluble AC activity, leading to increased cAMP levels. Interaction of this mechanism with the reversely directed effect of GPR81 on the cAMP levels in neurons and astrocytes may serve to stabilize synaptic pH. In turn, this effect may tell the neurons to spare their glucose and glycogen when external glycolytic substrates are available. Thus there are several rationales for the here observed concentration of GPR81 at the synaptic membrane, where it co-localizes with the lactate transporter MCT2 and glutamate receptors (Bergersen et al. 2001, 2005).
cAMP is known to downregulate MCTs by endocytotic internalization from the plasma membrane, for example, in cerebrovascular endothelial cells (Smith et al. 2012) and in intestinal epithelial cells (Borthakur et al. 2012). In the latter case, the surface density of MCT1 is enhanced in response to nutrient availability through lowering of cAMP by GPR109A (HCA2, a butyrate and nicotinate receptor), a close homologue of GPR81 (HCA1). An analogous regulation of MCTs on brain cells may be exerted by lactate through GPR81. Other proteins, such as glutamate receptors (Middei et al. 2013) and glucose transporters (e.g., Kim et al. 2012) that are subject to regulations through mechanisms involving the cAMP/CREB pathway, may be similarly influenced by GPR81.
An intriguing additional possibility, suggested by the coexistence of GPR81 with MCTs on subplasmalemmal vesicular organelles, is that cytosolic lactate can reach the active site of GPR81 on the inside of the vesicular organelles and thereby exert regulatory functions.
In conclusion, the present study identifies a cerebral receptor, GPR81 (HCA1), which allows lactate to serve as a volume transmitter, linking neurotransmission, neurovascular coupling, and brain energy metabolism.
This study was supported by research grants from The Research Council of Norway and The Lundbeck Foundation, Denmark, and The South-Eastern Norway Regional Health Authority.
We thank Vidar Gundersen at the University of Oslo for advice. We also thank Stefan Offermanns and Sorin Tunaru at the Max-Planck Institute for Heart and Lung Research, Bad Nauheim, Germany, and Eric Kalkhoven at the UMC Utrecht, The Netherlands, for providing GPR81 plasmids. Conflict of Interest: None declared.