PHD1 controls muscle mTORC1 in a hydroxylation-independent manner by stabilizing leucyl tRNA synthetase methods
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PHD1 levels and LRS activity decline during aging
Aging is associated with a loss of muscle mass. Moreover, the ability of older muscle to efficiently activate protein synthesis in response to amino acids, a condition termed anabolic resistance, is impaired -. Several molecular mechanisms underlying anabolic resistance have been proposed, including reduced amino acid delivery and uptake into the muscle, but it is not known whether leucine sensing mechanisms are affected. Thus, to explore the relevance of our findings in a human setting, we compared PHD1 protein content in muscle samples from a small cohort where samples were obtained from healthy old ( n = 8, 4 women and 4 men, 72.6 ± 2.3 y (mean ± SEM)) vs. young ( n = 8, 4 women and 4 men, 26.1 ± 1.1 y) volunteers after an overnight fast. In this cohort, old people showed reduced activ...
PHD1 controls muscle mass in a cell-autonomous fashion
We next confirmed that loss of Phd1 in muscle suffices to blunt the activation of mTORC1 downstream targets following leucine but not insulin stimulation (Fig. and Supplementary Fig. ). Moreover, leucine-induced protein synthesis assessed via puromycin incorporation was reduced as well (Supplementary Fig. ). To further confirm the cell autonomous role of PHD1 in the control of leucine mediated mTORC1 activation, we isolated muscle stem cells from WT and PHD1 KO mice and differentiated them to myotubes. Myotubes were starved of amino acids and stimulated with leucine. In WT myotubes, leucine dose dependently activated mTORC1, and maximal activation was reached at 5 mM (Fig. ). Loss of Phd1 reduced the mTORC1 response at all leucine concentrations tested (Fig. ). Off note, we also measured lower mTORC1 activity under full medium conditions (containing...
Methods
Isolation and culture of primary MPs: Muscle tissue was digested in HBSS supplemented with 1.5% bovine serum albumin (BSA) and 2 mg/mL collagenase type II (ThermoFisher Scientific, 17101015) for 1 h at 37 °C. After centrifugation, the cell pellet was then filtered using 40 and 100 µm cell strainers and a heterogeneous cell population was purified by FACS sorting or by serial preplating. For FACS, MPs were sorted based on positive alpha 7-integrin (1:100) and absence of Sca1, CD31, and CD45 (1:1000, see Supplementary Table ). For serial preplating, a freshly extracted heterogeneous cell population was preplated for 1.5 h at day 0, 4, and 10 after cell extraction. MPs were cultured in growth medium (see above) on dishes coated with Matrigel Basement Membrane Matrix (Corning, #356237, 1/25 dilution). When cells reached 80% confluency, growth...
Methods
Leucine stimulation: differentiated myotubes were starved for 1 h in low-glucose DMEM lacking all amino acids (US Biology, D9800-13), supplemented with 10% dialyzed FBS (dFBS) (ThermoFisher Scientific, 26400-044). Leucine was supplemented for 30 min in the indicated concentrations. For the RagA leucylation experiments, differentiated myotubes were incubated with DMEM containing all AA, except leucine (Sigma, 9443) + 10% FBS for 16 h supplemented with 0, 0.8, or 5 mM leucine.
In vitro pull-down assay
HEK293T were transfected with Myc-LRS and flag-PHD1 for 72 h and were subsequently lysed (50 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM MgCl2, 0.1% CHAPS and 0.5% Triton X-100 supplemented with protease inhibitors). Cell lysates were centrifuged at 15,000 g for 20 min. A total of 300 µg protein from the supernatant fraction was incubated with ANTI-FLAG ® M2 Affinity Gel (Sigma, A2220-1ML) at 4 °C overnight. After washing 3 times in lysis buffer supplemented with 50 mM NaCl, the precipitates were dissolved in laemmli buffer.
Animals
Whole body Phd1 knockout (PHD1 KO ) mice (50% Swiss/50% 129S1 background) were previously generated. Phd1 fl/fl mice were generated using homologous recombination in embryonic stem (ES) cells. The targeting vector was built in a pPNTlox2 plasmid with loxP sites flanking the neomycine resistance (neor) cassette, and contained from 5′ to 3′: a 5.3-kb EcoRV-HindIII fragment comprising exon 2 and the majority of intron 2 as 5′ homology arm, a 1.8-kb floxed neor cassette, a 2.5-kb HindIII-EcoRV fragment comprising exon 3 through 6, in which a third loxP site was introduced downstream of exon 4 along with a novel EcoRI site for genotyping purposes, an SV40 thymidine kinase expression cassette for negative selection purposes. The introduced third loxP site together with the loxP site located 3′ of the neor cassette thus flanked the Phd1 gene segment comprising exon 3...
Immunohistochemistry and histology
Fiber typing: skeletal muscle sections (10 µm) were dried and washed for 5 min in phosphate-buffered saline (PBS) supplemented with 0.05% triton (PBST) and subsequently blocked for 60 min in PBST + 10% goat serum (ThermoFisher Scientific, 16200-064). Afterwards a primary antibody cocktail was applied for 120 min for myosin heavy chain I (1:50), IIa (1:200) and b (1:100) (see Supplementary Table ) diluted in PBST + 10% goat serum. After washing 3 times for 5 min, a secondary antibody cocktail, diluted in PBST + 10% goat serum, was applied for goat anti-mouse Alexa Fluor 488,350,568 and wheat germ agglutinin Alexa fluor 647 (1:250) (ThermoFisher Scientific, see Supplementary Table ) for 60 min. Slides were mounted after a 3 × 5 min wash, sealed with glass cover slips and imag...
Immunohistochemistry and histology
LAMP2-mTORC1 colocalization: WT and PHD1 KO myoblasts were differentiated in matrigel-coated (1:25) 8-well chambers (ibidi; 80826). Briefly, fully differentiated myotubes were untreated, starved for 1 h in HBSS or starved in HBSS for 1 h and restimulated with differentiation medium for 1 h, after which they were rinsed with ice-cold (4 °C) PBS once and fixed for 5 min with 4% paraformaldehyde in PBS. The chamber wells were rinsed twice with PBS and permealized with 0.2% Triton X-100 in PBS for 15 min. After rinsing twice with PBS, the wells were blocked for one hour in blocking buffer (0.25% BSA in PBS) and incubated with primary antibody for LAMP2 and mTOR (1:200) in blocking buffer overnight at 4 °C (see Supplementary Table ). Subsequently, the wells were rinsed twice with blocking buffer and incubated with secondary antibodi...
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Evidence quotes (8)
Aging is associated with a loss of muscle mass. Moreover, the ability of older muscle to efficiently activate protein synthesis in response to amino acids, a condition termed anabolic resistance, is impaired -. Several molecular mechanisms underlying anabolic resistance have been proposed, including reduced amino acid delivery and uptake into the muscle, but it is not known whether leucine sensing mechanisms are affected. Thus, to explore the relevance of our findings in a human setting, we compared PHD1 protein content in muscle samples from a small cohort where samples were obtained from healthy old ( n = 8, 4 women and 4 men, 72.6 ± 2.3 y (mean ± SEM)) vs. young ( n = 8, 4 women and 4 men, 26.1 ± 1.1 y) volunteers after an overnight fast. In this cohort, old people showed reduced activation of muscle protein synthesis in response to milk protein (containing a high dose of leucine) ingestion when compared to young subjects. We found that older muscle on average showed an almost 50% decrease in PHD1 levels (Fig. ). Lower PHD1 content was confirmed using immunofluorescent sta...
We next confirmed that loss of Phd1 in muscle suffices to blunt the activation of mTORC1 downstream targets following leucine but not insulin stimulation (Fig. and Supplementary Fig. ). Moreover, leucine-induced protein synthesis assessed via puromycin incorporation was reduced as well (Supplementary Fig. ). To further confirm the cell autonomous role of PHD1 in the control of leucine mediated mTORC1 activation, we isolated muscle stem cells from WT and PHD1 KO mice and differentiated them to myotubes. Myotubes were starved of amino acids and stimulated with leucine. In WT myotubes, leucine dose dependently activated mTORC1, and maximal activation was reached at 5 mM (Fig. ). Loss of Phd1 reduced the mTORC1 response at all leucine concentrations tested (Fig. ). Off note, we also measured lower mTORC1 activity under full medium conditions (containing 0.8 mM leucine), but consistent with our in vivo data, the response to insulin was preserved (Supplementary Fig. ).
Isolation and culture of primary MPs: Muscle tissue was digested in HBSS supplemented with 1.5% bovine serum albumin (BSA) and 2 mg/mL collagenase type II (ThermoFisher Scientific, 17101015) for 1 h at 37 °C. After centrifugation, the cell pellet was then filtered using 40 and 100 µm cell strainers and a heterogeneous cell population was purified by FACS sorting or by serial preplating. For FACS, MPs were sorted based on positive alpha 7-integrin (1:100) and absence of Sca1, CD31, and CD45 (1:1000, see Supplementary Table ). For serial preplating, a freshly extracted heterogeneous cell population was preplated for 1.5 h at day 0, 4, and 10 after cell extraction. MPs were cultured in growth medium (see above) on dishes coated with Matrigel Basement Membrane Matrix (Corning, #356237, 1/25 dilution). When cells reached 80% confluency, growth medium was switched to differentiation medium containing low-glucose DMEM, 2% HS and 1% P/S. MPs were fully differentiated after 3 days.
Leucine stimulation: differentiated myotubes were starved for 1 h in low-glucose DMEM lacking all amino acids (US Biology, D9800-13), supplemented with 10% dialyzed FBS (dFBS) (ThermoFisher Scientific, 26400-044). Leucine was supplemented for 30 min in the indicated concentrations. For the RagA leucylation experiments, differentiated myotubes were incubated with DMEM containing all AA, except leucine (Sigma, 9443) + 10% FBS for 16 h supplemented with 0, 0.8, or 5 mM leucine.
HEK293T were transfected with Myc-LRS and flag-PHD1 for 72 h and were subsequently lysed (50 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM MgCl2, 0.1% CHAPS and 0.5% Triton X-100 supplemented with protease inhibitors). Cell lysates were centrifuged at 15,000 g for 20 min. A total of 300 µg protein from the supernatant fraction was incubated with ANTI-FLAG ® M2 Affinity Gel (Sigma, A2220-1ML) at 4 °C overnight. After washing 3 times in lysis buffer supplemented with 50 mM NaCl, the precipitates were dissolved in laemmli buffer.
Whole body Phd1 knockout (PHD1 KO ) mice (50% Swiss/50% 129S1 background) were previously generated. Phd1 fl/fl mice were generated using homologous recombination in embryonic stem (ES) cells. The targeting vector was built in a pPNTlox2 plasmid with loxP sites flanking the neomycine resistance (neor) cassette, and contained from 5′ to 3′: a 5.3-kb EcoRV-HindIII fragment comprising exon 2 and the majority of intron 2 as 5′ homology arm, a 1.8-kb floxed neor cassette, a 2.5-kb HindIII-EcoRV fragment comprising exon 3 through 6, in which a third loxP site was introduced downstream of exon 4 along with a novel EcoRI site for genotyping purposes, an SV40 thymidine kinase expression cassette for negative selection purposes. The introduced third loxP site together with the loxP site located 3′ of the neor cassette thus flanked the Phd1 gene segment comprising exon 3 and 4, which encode part of the catalytic domain conferring the prolyl hydroxylase activity. The construct was linearized with NotI and electroporated into G4 ES cells of 129SvEv/C57BL6 origin (kind gift from A. Nagy, Toronto). After positive-negative drug selection with 200 µg/ml...
Fiber typing: skeletal muscle sections (10 µm) were dried and washed for 5 min in phosphate-buffered saline (PBS) supplemented with 0.05% triton (PBST) and subsequently blocked for 60 min in PBST + 10% goat serum (ThermoFisher Scientific, 16200-064). Afterwards a primary antibody cocktail was applied for 120 min for myosin heavy chain I (1:50), IIa (1:200) and b (1:100) (see Supplementary Table ) diluted in PBST + 10% goat serum. After washing 3 times for 5 min, a secondary antibody cocktail, diluted in PBST + 10% goat serum, was applied for goat anti-mouse Alexa Fluor 488,350,568 and wheat germ agglutinin Alexa fluor 647 (1:250) (ThermoFisher Scientific, see Supplementary Table ) for 60 min. Slides were mounted after a 3 × 5 min wash, sealed with glass cover slips and imaged with a epifluorescent microscope (Zeiss Axio observer Z.1) at 10 ×. Fiber cross-sectional area was automatically determined with a Muscle J plugin for Image J software. Muscle fiber type was manually counted on tile scans. To determine the presence of centrally nucleated fibers, sections wer...
LAMP2-mTORC1 colocalization: WT and PHD1 KO myoblasts were differentiated in matrigel-coated (1:25) 8-well chambers (ibidi; 80826). Briefly, fully differentiated myotubes were untreated, starved for 1 h in HBSS or starved in HBSS for 1 h and restimulated with differentiation medium for 1 h, after which they were rinsed with ice-cold (4 °C) PBS once and fixed for 5 min with 4% paraformaldehyde in PBS. The chamber wells were rinsed twice with PBS and permealized with 0.2% Triton X-100 in PBS for 15 min. After rinsing twice with PBS, the wells were blocked for one hour in blocking buffer (0.25% BSA in PBS) and incubated with primary antibody for LAMP2 and mTOR (1:200) in blocking buffer overnight at 4 °C (see Supplementary Table ). Subsequently, the wells were rinsed twice with blocking buffer and incubated with secondary antibodies (diluted in blocking buffer 1:500) for one hour at room temperature in dark. Individual myotubes were imaged with 60 × confocal microscope (Olympus olympus fluoview FV 3000). LAMP2-mTOR colocalization was determined by ImageJ using a plugin described by Moser et al.. This analysis combines o...
Machine-readable layer
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"text": "We next confirmed that loss of Phd1 in muscle suffices to blunt the activation of mTORC1 downstream targets following leucine but not insulin stimulation (Fig. and Supplementary Fig. ). Moreover, leucine-induced protein synthesis assessed via puromycin incorporation was reduced as well (Supplementary Fig. ). To further confirm the cell autonomous role of PHD1 in the control of leucine mediated mTORC1 activation, we isolated muscle stem cells from WT and PHD1 KO mice and differentiated them to myotubes. Myotubes were starved of amino acids and stimulated with leucine. In WT myotubes, leucine dose dependently activated mTORC1, and maximal activation was reached at 5 mM (Fig. ). Loss of Phd1 reduced the mTORC1 response at all leucine concentrations tested (Fig. ). Off note, we also measured lower mTORC1 activity under full medium conditions (containing..."
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