Exercise induces cerebral VEGF and angiogenesis via the lactate receptor HCAR1 methods
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Lactate injections increase brain angiogenesis
Interestingly, the increased vascularization in wild-type mice, and the lack of effect in Hcar1 knockouts, was reproduced by daily subcutaneous injections of sodium L-lactate (2 g kg -1 bodyweight; 200 mg ml -1; pH 7.4; that is, 18 mmol kg -1; raising blood lactate to ∼10 mM; ) 5 days a week for 7 weeks ( ). As lactate injections have been observed to cause acute anxiety attacks in susceptible individuals, the mice were tested 15 min after the first lactate injection in a rodent test for anxiety, the 'elevated zero maze' (see Methods), to rule out the possibility that the results were confounded by anxiety; the test showed no difference between the groups ( ). We therefore conclude that lactate acting on HCAR1 is a pivotal regulator of angiogenesis in the brain and underlies the angiogenic effect of exerc...
Animal treatment
All mice were 7-9 weeks of age at the start of the experiments. Hcar1 knockout or wild-type mice (both sexes with about equal M/F distribution, see ) were randomized into three groups: treadmill running, sodium L-lactate injections or saline injections (control). The mice that were treated with lactate received a subcutaneous injection of sodium L-lactate (≥99.0%, Aldrich, 71718; 2 g kg -1 bodyweight; 200 mg ml -1 dissolved in 0.9% saline; pH-adjusted to 7.4; that is, 18 mmol kg -1 ). The control mice received the same volume (per kg bodyweight) of 0.9% saline. The lactate or saline injections were administered subcutaneously 5 days a week for 7 weeks. The mice were weighed every week for adjustment of the dose.
Animal treatment
The interval exercise regime has been previously described. It is designed for optimum gain in cardiovascular function and to reach ∼90% of VO 2max during the high-intensity intervals. Briefly, each session consisted of 10 min warm-up at 5 m min -1, followed by 10 high-intensity intervals of 4 min each, and separated by 2 min of active rest. Running took place on a treadmill (Columbus Instruments, USA) at a 25 degrees incline. The mice were exposed to the high-intensity interval exercise protocol for 5 consecutive days each week, for a total duration of 7 weeks. On the first day of the exercise intervention, and then every other week ( ), a maximal exercise-capacity test was performed for each individual mouse, to adjust the running speed of the training intervals to near the maximum they could sustain during 10 consecutive intervals. After...
Plasma lactate measurements
For plasma lactate measurements, the mice were anaesthetized mildly with isoflurane and decapitated at exactly 5, 15, 30, 60 or 180 min after the dose of lactate ( n =5 for each time point) or saline ( n =3 for each time point). The blood was collected into 0.5 ml Minicollect tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) containing 2.5 mg sodium fluoride (NaF) and 2.0 mg potassium oxalate (KOx) per ml to prevent post vivo glycolysis (with the formation of lactate) and coagulation, respectively. Blood samples were centrifuged, and the resulting plasma samples were frozen in liquid nitrogen and stored at -80 °C. The lactate measurements were performed using VITROS DT60II and VITROS LAC DT slides (Ortho Clinical Diagnostics, UK). The plasma samples were diluted 1:1 with water to ensure that all samples were below the maximum detection...
Quantitative western blotting
At 6 h after the end of the exercise, or 6 h after the last dose of L-lactate or saline, the mice were deeply anaesthetized with isoflurane and killed by decapitation. The hippocampi were carefully dissected out on ice and snap-frozen in liquid nitrogen. Material from in vitro experiments was processed similarly. For western blot analysis, lysates were prepared by homogenizing in radio-immunoprecipitation assay buffer (Sigma-Aldrich) containing cOmplete Protease Inhibitor Cocktail (Roche). Samples, 10 µg of total protein, were subjected to gel electrophoresis (12% SDS/PAGE (Bio-Rad)), transferred to a nitrocellulose membrane, using Transblot Turbo (Bio-Rad) and incubated overnight with the following primary antibodies: rabbit anti-VEGFA (ab46154; Abcam; diluted 1:1,000), mouse anti-α-tubulin (3873S; Cell Signaling Technologies (CST), Beverly, MA, USA; dilut...
Immunohistochemistry and fluorescence microscopy
At 6 h after the end of the exercise or 6 h after the last dose of lactate/saline, Hcar1 knockout and wild-type mice were deeply anaesthetized with zolazepam 3.3 mg, tiletamine 3.3 mg, xylazine 0.5 mg, fentanyl 2.6 µg ml -1; 0.1 ml 10 g -1 bodyweight, intraperitoneally (i.p.) and transcardially perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4 (NaPi) for 8 min. Non-treated Hcar1 knockout and wild-type mice and mRFP-HCAR1 reporter mice were anaesthetized and perfused in a similar manner. Brains from some mRFP reporter mice were viewed without perfusion or further processing ( ). After perfusion fixation, organs were gently removed, viewed with a stereomicroscope (for example, ), and stored in fixative at 4 °C until cutting 50-µm-thick vibratome sections or 20-_...
HCAR1 immunocytochemistry
Mice (wild-type, Hcar1 knockout and mRFP-HCAR1 reporter) were anaesthetized and transcardially perfused with 4% paraformaldehyde in PBS before brain removal and post fixation in the same fixative for 24 h (ref. ). Following dehydration in ethanol and embedding in paraffin, 4 µm thick sagittal sections through the entire forebrain were cut with a microtome (Thermo Scientific). Test sections were deparaffinized in Neoclear (Millipore) followed by rehydration through an ethanol gradient (100%, 3 min; 100%, 3 min; 96%; 1 min and 70% 1 min) and subsequently incubated at 100 °C, under pressure of 0.8 -1 bar in citrate antigen retrieval buffer (pH 6.0), containing 0.05% Tween 20 (Sigma) for 2 min. Test sections were then washed in Milli-Q water and PBS containing 0.1% Tween 20. Additional sagittal brain sections derived f...
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Evidence quotes (7)
Interestingly, the increased vascularization in wild-type mice, and the lack of effect in Hcar1 knockouts, was reproduced by daily subcutaneous injections of sodium L-lactate (2 g kg -1 bodyweight; 200 mg ml -1; pH 7.4; that is, 18 mmol kg -1; raising blood lactate to ∼10 mM; ) 5 days a week for 7 weeks ( ). As lactate injections have been observed to cause acute anxiety attacks in susceptible individuals, the mice were tested 15 min after the first lactate injection in a rodent test for anxiety, the 'elevated zero maze' (see Methods), to rule out the possibility that the results were confounded by anxiety; the test showed no difference between the groups ( ). We therefore conclude that lactate acting on HCAR1 is a pivotal regulator of angiogenesis in the brain and underlies the angiogenic effect of exercise.
All mice were 7-9 weeks of age at the start of the experiments. Hcar1 knockout or wild-type mice (both sexes with about equal M/F distribution, see ) were randomized into three groups: treadmill running, sodium L-lactate injections or saline injections (control). The mice that were treated with lactate received a subcutaneous injection of sodium L-lactate (≥99.0%, Aldrich, 71718; 2 g kg -1 bodyweight; 200 mg ml -1 dissolved in 0.9% saline; pH-adjusted to 7.4; that is, 18 mmol kg -1 ). The control mice received the same volume (per kg bodyweight) of 0.9% saline. The lactate or saline injections were administered subcutaneously 5 days a week for 7 weeks. The mice were weighed every week for adjustment of the dose.
The interval exercise regime has been previously described. It is designed for optimum gain in cardiovascular function and to reach ∼90% of VO 2max during the high-intensity intervals. Briefly, each session consisted of 10 min warm-up at 5 m min -1, followed by 10 high-intensity intervals of 4 min each, and separated by 2 min of active rest. Running took place on a treadmill (Columbus Instruments, USA) at a 25 degrees incline. The mice were exposed to the high-intensity interval exercise protocol for 5 consecutive days each week, for a total duration of 7 weeks. On the first day of the exercise intervention, and then every other week ( ), a maximal exercise-capacity test was performed for each individual mouse, to adjust the running speed of the training intervals to near the maximum they could sustain during 10 consecutive intervals. After a 15 min warm-up period at 9.6 m min -1, the band speed was increased by 1.8 m min -1 every 2 min until exhaustion, that is, the mice refused to run further, despite being manually placed back on to the band or receiving electrical stimuli (maximally...
For plasma lactate measurements, the mice were anaesthetized mildly with isoflurane and decapitated at exactly 5, 15, 30, 60 or 180 min after the dose of lactate ( n =5 for each time point) or saline ( n =3 for each time point). The blood was collected into 0.5 ml Minicollect tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) containing 2.5 mg sodium fluoride (NaF) and 2.0 mg potassium oxalate (KOx) per ml to prevent post vivo glycolysis (with the formation of lactate) and coagulation, respectively. Blood samples were centrifuged, and the resulting plasma samples were frozen in liquid nitrogen and stored at -80 °C. The lactate measurements were performed using VITROS DT60II and VITROS LAC DT slides (Ortho Clinical Diagnostics, UK). The plasma samples were diluted 1:1 with water to ensure that all samples were below the maximum detection limit of the method (15 mM). All samples were analysed in duplicates.
At 6 h after the end of the exercise, or 6 h after the last dose of L-lactate or saline, the mice were deeply anaesthetized with isoflurane and killed by decapitation. The hippocampi were carefully dissected out on ice and snap-frozen in liquid nitrogen. Material from in vitro experiments was processed similarly. For western blot analysis, lysates were prepared by homogenizing in radio-immunoprecipitation assay buffer (Sigma-Aldrich) containing cOmplete Protease Inhibitor Cocktail (Roche). Samples, 10 µg of total protein, were subjected to gel electrophoresis (12% SDS/PAGE (Bio-Rad)), transferred to a nitrocellulose membrane, using Transblot Turbo (Bio-Rad) and incubated overnight with the following primary antibodies: rabbit anti-VEGFA (ab46154; Abcam; diluted 1:1,000), mouse anti-α-tubulin (3873S; Cell Signaling Technologies (CST), Beverly, MA, USA; diluted 1:1,000), mouse anti-ERK1/2 (anti-p44/42 MAPK, 4696S; CST; diluted 1:1,000), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204; anti-phospho-p44/42 MAPK, 4370; CST, diluted 1:1,000), mouse anti-Akt (pan; 4691P; CST; diluted 1:1,000), rabbit anti-phospho-Act (Ser473; 4060S; CST, diluted 1:1,000). Protein...
At 6 h after the end of the exercise or 6 h after the last dose of lactate/saline, Hcar1 knockout and wild-type mice were deeply anaesthetized with zolazepam 3.3 mg, tiletamine 3.3 mg, xylazine 0.5 mg, fentanyl 2.6 µg ml -1; 0.1 ml 10 g -1 bodyweight, intraperitoneally (i.p.) and transcardially perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4 (NaPi) for 8 min. Non-treated Hcar1 knockout and wild-type mice and mRFP-HCAR1 reporter mice were anaesthetized and perfused in a similar manner. Brains from some mRFP reporter mice were viewed without perfusion or further processing ( ). After perfusion fixation, organs were gently removed, viewed with a stereomicroscope (for example, ), and stored in fixative at 4 °C until cutting 50-µm-thick vibratome sections or 20-µm-thick frozen sections (after cryoprotection by immersion in 30% sucrose in 0.1 M NaPi solution overnight). Free floating brain sections were washed three times in PBS(10 mM NaPi, 0.9% NaCl) and then subjected to fluorescence immunocytochemistry, directly, or after being incubated for...
Mice (wild-type, Hcar1 knockout and mRFP-HCAR1 reporter) were anaesthetized and transcardially perfused with 4% paraformaldehyde in PBS before brain removal and post fixation in the same fixative for 24 h (ref. ). Following dehydration in ethanol and embedding in paraffin, 4 µm thick sagittal sections through the entire forebrain were cut with a microtome (Thermo Scientific). Test sections were deparaffinized in Neoclear (Millipore) followed by rehydration through an ethanol gradient (100%, 3 min; 100%, 3 min; 96%; 1 min and 70% 1 min) and subsequently incubated at 100 °C, under pressure of 0.8 -1 bar in citrate antigen retrieval buffer (pH 6.0), containing 0.05% Tween 20 (Sigma) for 2 min. Test sections were then washed in Milli-Q water and PBS containing 0.1% Tween 20. Additional sagittal brain sections derived from Hcar1 knockout mice were prepared as described above and used to pre-absorb the primary antibody solution, containing anti-HCAR1 antibody (SAB1300090 SIGMA anti-mouse Gpr81-s296 antibody produced in rabbit, affinity-isolated; 1:200 dilution), 10% goat serum, 10% BSA and 0.1% Tween 20 for 1...
Machine-readable layer
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