Download Lactate Receptor Sites Link Neurotransmission

Cerebral Cortex October 2014;24:2784–2795
doi:10.1093/cercor/bht136
Advance Access publication May 21, 2013
Lactate Receptor Sites Link Neurotransmission, Neurovascular Coupling,
and Brain Energy Metabolism
Knut H. Lauritzen1,3,4, Cecilie Morland1,2, Maja Puchades2, Signe Holm-Hansen1,3,4, Else Marie Hagelin5, Fredrik Lauritzen1,3,4,
Håvard Attramadal5,6, Jon Storm-Mathisen1,2, Albert Gjedde3,4 and Linda H. Bergersen1,3,4,7
1
The Brain and Muscle Energy Group, 2Glio- and Neurotransmitter Group, Synaptic Neurochemistry Lab, Department of Anatomy
and Centre for Molecular Biology and Neuroscience/SERTA Healthy Brain Aging, Institute of Basic Medical Sciences, University of
Oslo, Oslo, Norway, 3Department of Neuroscience and Pharmacology, 4Center for Healthy Aging, Faculty of Health Sciences,
University of Copenhagen, Copenhagen, Denmark, 5Institute for Surgical Research, Oslo University Hospital, Oslo, Norway,
6
Center for Heart Failure Research and 7Institute of Oral Biology, University of Oslo, Norway
Address correspondence to Dr Linda H. Bergersen, The Brain and Muscle Energy Group, Department of Anatomy, Institute of Basic Medical
Sciences, University of Oslo, P.O. Box 1105 Blindern, NO-0317 Oslo, Norway. Email: [email protected]
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.
Keywords: astrocytic vascular end-feet, electron microscopy, excitatory
synapses, lactate, volume transmitter
Introduction
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© The Author 2013. Published by Oxford University Press. All rights reserved.
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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 Gicoupled 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
Animal Handling
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.
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
R 2-values <0.99 were rejected.
Immunoperoxidase Histochemistry
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 gelatinecoated 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.
Western Blotting
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).
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 RNaseFree 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 TaqManmix 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
Immunofluorescence Histochemistry
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
Anti-GPR81 Antibody
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.
Cerebral Cortex October 2014, V 24 N 10 2785
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-diamidino2-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 0.1 M 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 0.1 M 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, 0.05 M
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 (15 min) and
lead citrate (90 s), 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
Figure 1. Schematic illustration of brain subcellular structures examined. From left to right: An excitatory-type synapse between a presynaptic terminal and a dendritic spine
exhibiting an asymmetric specialization (i.e., with postsynaptic density, PSD), an astrocyte with perivascular end-foot, abluminal, and luminal membrane of the endothelial cell,
forming the blood–brain barrier. Analysis of gold particle density (number of gold particles per micron of membrane length) was performed in 7 specific membrane compartments
that were defined as: the postsynaptic membrane (red), postsynaptic perisynaptic membrane (violet blue), presynaptic membrane (blue), presynaptic perisynaptic membrane
(green), perivascular end-foot membrane ( pink), abluminal membrane (yellow), and luminal membrane (orange).
2786 Lactate Receptor Sites Link Neurotransmission, Neurovascular Coupling
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Lauritzen et al.
Figure 2. Immunohistochemical distribution of GPR81 in bran sagittal sections. (A) Diagram of a parasagittal section indicating the positions of the areas shown in panels B–H
(identified according to Franklin and Paxinos 2007). (B) Cerebellar cortex, Purkinje cell bodies (arrows), and dendrites (arrowheads) are strongly labeled. (C) CA3 of hippocampus,
pyramidal neurons are stained. (D) Dentate area, staining of neurons, and dendrites, mainly in the dentate hilus. (E) Primary somato-motor area, layer IV, GPR81-positive pyramidal
cells. The other layers and other regions in the cerebral neocortex showed similar labeling patterns (cf G). (F) Striatum, neuronal labeling is weak. (G) Secondary visual cortex, low
power view, pyramidal cells are stained. (H) Negative control where the GPR81 antibody was omitted, processed, and photographed identically with G, cellular staining is absent.
Symbols: Arrows, cell bodies; arrowheads, dendrites; Cb, cerebellum; ml, molecular layer; Pcl, Purkinje cell layer; gcl, granular cell layer; Hc, hippocampus CA3; o, stratum oriens;
P, pyramidal cell layer; lu, stratum lucidum; r, stratum radiatum; Dg, dentate gyrus; gcl, granule cell layer; Mcx, primary motor cortex, M1; Vcx, secondary visual cortex, V2;
Str, striatum; wm, white matter. Scale bar: 25 µm.
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 proximodistal 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
Cerebral Cortex October 2014, V 24 N 10 2787
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 30 min at 30 °C. Subsequently, assay of cAMP generation was
initiated, and the slices were incubated for 16 min 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/).
Results
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
2788 Lactate Receptor Sites Link Neurotransmission, Neurovascular Coupling
•
Figure 3. Expression-profile of GPR81 mRNA in selected brain regions and adipose
tissue, and immunoblot of GPR81 in brain extract, adipose tissue and transfected HeLa
cells. (A) Quantitative RT-PCR data showing expression of GPR81 in the cerebellum
(Cb), hippocampus (Hc), and cerebral cortex (Cx). (B) Quantitative RT-PCR data
showing expression of GPR81 in omental adipose tissue (OAF). Value for Cb was set to
1, and all other regions compared with Cb. Data are expressed as mean + SEM.
(C) Immunoblots using the GPR81-S296 antibody show the presence of GPR81 protein
in mouse whole-brain and mouse omental adipose tissue (fat), as well as in HeLa cells
transfected with murine GPR81 (HeLa GPR81). A strong band at the predicted
molecular mass, 39 kDa (arrow), was detected in all samples and was the
predominant band in brain. Untransfected HeLa cells (HeLa) show only a faint band at
39 kDa, consistent with a slight possible endogenous GPR81 expression. All samples
show an additional band at ∼60 kDa, which probably represents a form of GPR81, as is
enhanced in the transfected HeLa-cells, possibly the glycosylated receptor. The lower
molecular mass bands could represent proteolytic fragments of the protein.
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
Lauritzen et al.
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). Doublelabeling 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 halfmaximal 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 (R 2 ≈ 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).
Discussion
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–
Cerebral Cortex October 2014, V 24 N 10 2789
Figure 4. GPR81 is present in neurons (A and C), in astrocytes (B and D) and at microvessels (B). Immunolabeling of coronal paraffin-embedded sections demonstrates the
presence of GPR81 in cellular elements in hippocampus CA1 (A and B) and cerebellum (C and D). MAP2 was used as a neuronal marker (A and C). Glutamine synthetase (GS) was
used as astrocyte marker (B and D). There is partial co-localization of GPR81 with either MAP2 or GS. In the hippocampus, GPR81 is concentrated in perikarya and proximal parts of
dendrites (A, arrows) of pyramidal cells, whereas MAP2 is concentrated in distal parts of dendrites. GPR81 is partly associated with GS-positive astrocytes (B, arrowheads), and
perivascular astrocytic end-feet (B, narrow arrowheads) can be seen outlining GRP81 stained structures, likely parts of endothelial cells, between the end-foot and the blood vessel
lumen. In the cerebellum, GPR81 is concentrated in Purkinje cell perikarya (identified by their size and localization), and in some neuronal structures, likely perikarya (C, long arrows)
or likely synaptic glomeruli (C, short arrows), in the granule cell layer. Some GS stained cerebellar astroglia are also stained for GPR81 (D, arrowheads). Sections were counterstained
with DAPI to show cell nuclei. Symbols: arrows, neuronal structures; arrowheads, astrocytic structures; o, stratum oriens; p, pyramidal cell layer; r, stratum radiatum; Pcl, Purkinje
cell layer; gl, granule cell layer. Scale bars: 20 µm.
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-neuronlactate shuttle, implying that lactate derived from astrocytes
can enter neurons where it serves as a substrate of oxidative
2790 Lactate Receptor Sites Link Neurotransmission, Neurovascular Coupling
•
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
Lauritzen et al.
Figure 5. Lactate receptor GRP81 in adipocytes (A), at the blood–brain interface (B, C, and D), and at brain excitatory synapses (E–I): Electron microscopic immunogold localization.
(A) Electron micrograph of adipocytes from rat omental adipose tissue. (B) Electron micrograph of vascular endothelium and perivascular astrocytic end-foot at a hippocampal blood
vessel. (C–F) Quantification of GPR81 immunoreactivity in blood vessel endothelium and astrocytic vascular end-feet in hippocampus CA1 stratum radiatum (C) and cerebellar
molecular layer (D), and in excitatory synapses in the same regions of hippocampus (E) and cerebellum (F), see Figure 1 for membrane compartments. Data represent mean
values + SD of 60 synapses and 25–27 blood vessels in 3 mice. Particles within 30 nm on each side of the membrane midline were recorded, in accordance with the resolution of
the immunogold method. Background (Bg) represents average tissue immunoreactivity estimated as particles within 30 nm of lines drawn randomly across the observed sections
(20). (G–H) Electron micrographs showing localization of GPR81 (gold particles) in the synapse of Schaffer collateral/commissural fiber synapses in CA1 stratum radiatum (G) and
the parallel fiber–Purkinje cells in cerebellum (H). Note that most of the immunogold particles are in close proximity to the post- and presynaptic membranes, identified by the
presence of the postsynaptic density (PSD, black arrowheads), and to pre- and postsynaptic vesicular organelles. (I) Frequency plots for the proximo-distal distribution of GPR81 as a
function of the distance from the postsynaptic membrane at excitatory synapses in hippocampus and cerebellum (black and gray, respectively, 20 of each). The distance from the
center of the gold particle to the external face of the postsynaptic membrane, identified by the PSD area, was recorded perpendicular to the PSD, the positive values representing
particles located in postsynaptic direction. The average width of the synaptic cleft is 20 nm, corresponding to the bins marked −10 and −20 nm. Designations: Am, adipocyte
membrane; collagen, collagen fibrils in connective tissue extracellular space; L, luminal membrane of endothelial cell facing blood vessel lumen; Abl, abluminal membrane of
endothelial cell; Bg, “background” assessed as average immunogold particle density along randomly drawn 500-nm long lines avoiding mitochondria and cell membranes; Bl, basal
lamina (fused laminae of blood vessel and nervous tissue); Ef, end-foot membrane of astrocyte abutting on basal lamina; Pre, presynaptic plasma membrane; Post, postsynaptic
plasma membrane; PPost, perisynaptic membrane postsynaptically; PPre, perisynaptic membrane presynaptically; red arrowheads, 10 nm immunogold particles representing GPR81
(arrowheads pointing from extracellular aspect of cell indicate particles on the surface membrane, arrowheads pointing from intracellular aspect of cell indicate particles on
intracellular vesicular organelles); black arrowheads, borders of the PSDs; s, dendritic spine or thin dendritic branch; t, nerve terminal. *, statistically significant difference compared
with Bg (average particle density on section, P < 0.01, ANOVA); no statistically significant differences were obtained between hippocampus and cerebellum. Scale bars: A, 500 nm;
B, 100 nm; G, 100 nm.
Cerebral Cortex October 2014, V 24 N 10 2791
Figure 6. Double labeling for lactate receptor GRP81 and astrocyte marker GS at 2 excitatory type synapses (A and B) in hippocampus CA1 stratum radiatum. Note that in addition
to a large number of immunogold particles associated with the post- and presynaptic membranes and with pre- and postsynaptic vesicular organelles, a few particles (black
arrowheads) appear to be associated with the astrocytic plasma membrane. Designations: s, dendritic spine; t, nerve terminal; astrocyte, astrocytic perisynaptic process; red
arrowhead, 20-nm immunogold particle indicating GS; black arrowhead, 10-nm immunogold particle indicating GPR81 at astrocyte membrane. Scale bars: 100 nm.
Figure 7. Concentration-effect curves for GPR81 activation in slices of hippocampal
cortex in vitro. L-Lactate (▾) and 3,5-DHBA (▪) elicit concentration dependent inhibition
of forskolin (10 μM)-stimulated cAMP synthesis. Data are presented as percent of
forskolin-stimulated cAMP and are mean ± SEM of n = 4 independent experiments.
The concentrations of L-lactate employed do not cause complete inhibition of
forskolin-stimulated cAMP. The extrapolated curve for L-lactate models the effects of
higher concentrations of L-lactate assuming full agonist effect of L-lactate.
3,5-DHBA-stimulated inhibition of cAMP synthesis revealed a sigmoidal curve fit
(coefficient of determination R 2 ≈0.94; Hill slope ≈0.8) with IC50 at 1.4 mM (95%
confidence interval 0.58–3.3 mM). L-Lactate-stimulated inhibition of cAMP generation
also displayed sigmoidal curve fit with R 2≈0.89. The extrapolated curve for
L-lactate-stimulated inhibition of cAMP synthesis indicates an IC50 of ∼29 mM.
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,
2792 Lactate Receptor Sites Link Neurotransmission, Neurovascular Coupling
•
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
Lauritzen et al.
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 catecholaminestimulated 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 bicarbonateresponsive 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.
Funding
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.
Notes
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.
Cerebral Cortex October 2014, V 24 N 10 2793
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