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Macrophage-derived glutamine boosts satellite cells and muscle regeneration

Muscle regeneration is sustained by infiltrating macrophages and consequent satellite cell (SC) activation1–4. Macrophages and SC communicate in different ways1–5 but their metabolic interplay was never investigated so far. Here, we found that muscle injuries and aging are characterized by intratissutal glutamine restriction. Low glutamine levels endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity at the expense of glutamate dehydrogenase-1 (GLUD1)-mediated glutamine oxidation. Glud1 knockout (KO) macrophages display constitutively high GS activity which prevents glutamine shortage. Import of macrophage-derived glutamine by SC through the glutamine-transporter SLC1A5 activates mTOR and promotes SC proliferation and differentiation. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischemia, or aging. Conversely, SLC1A5 blockade in SC or GS inactivation in macrophages negatively affects SC functions and muscle regeneration. These results highlight a metabolic cross-talk between SC and macrophages whereby macrophage-derived glutamine sustains SC functions. Thus, GLUD1 targeting offers new therapeutic opportunities for the regeneration of injured or aged muscles.

Given the important role of glutamine in muscle homeostasis [9][10][11] , and our observation that glutamine production by macrophages remodels the composition of the extracellular tumormilieu 12,13 , we hypothesized a function of glutamine in a yet unidentified, metabolic crosstalk between macrophages and SC.
To induce myofiber death, inflammation and muscle regeneration, we injected cardiotoxin (CTX) in the tibialis anterior (TA) 14 or provoked ischemia of the crural muscles 15 in control (CTRL) and Glud1 ΔMo mice, with complete GLUD1 deletion in macrophages and only 38% knockdown in neutrophils (Extended Data Fig. 1a-c). CTRL and Glud1 ΔMo mice revealed similar muscle histology in healthy conditions and early after damage, i.e. 1 day post-CTX or 3 days post-femoral-artery-ligation ( Fig. 1a-e). However, compared to CTRL, Glud1 ΔMo mice displayed an earlier peak in the number of regenerating myofibers and a quicker resolution of muscle necrosis, cell death, oxidative damage, and inflammation ( Fig. 1a-m). Six days post-CTX, muscle viability was higher in Glud1 ΔMo vs. CTRL mice. Yet, the early regenerating myofibers (expressing embryonic myosin heavy chain) were fewer, but the late ones (negative for embryonic myosin heavy chain) were larger in Glud1 ΔMo mice, pointing to a faster and more advanced regeneration (Fig. 1n-p). This phenotype was due to monocyte-derived macrophages, rather than tissue-resident macrophages (Extended Data Fig. 1d,e). Inducible deletion of Glud1 in macrophages only led to improved muscle recovery as well (Extended Data Fig. 1f-i).
In voluntary wheel running tests, the baseline physical activity and its drop 1 day post-CTX were comparable in both genotypes. However, Glud1 ΔMo mice re-gained the pre-injured physical capabilities earlier than CTRL mice (Fig. 1q).
Basal numbers of SC, assessed by Pax7 expression 16 , was comparable between genotypes ( Fig. 1r-v). However, after injury, induction of SC proliferation (assessed by PHH3, or Ki67) and differentiation (assessed by the early and late differentiation markers MyoD and Myogenin, respectively 17 ) were stronger and quicker in Glud1 ΔMo than in CTRL mice (Fig. 1r-x; Extended Data Fig. 1j-n). Thus, muscle regeneration in Glud1 ΔMo mice is more efficient.
We then assessed how GLUD1-deficiency affects macrophage-mediated immunomodulation and vessel growth. Blood count, immune landscapes and vascular features of muscles at baseline and early after damage were similar in Glud1 ΔMo vs. CTRL mice (Extended Data Table1; Extended Data Fig. 1o-w). In vitro and in vivo recruitment assays, macrophage polarization, as well as wound healing and angiogenic functions did not change (Extended Data Fig. 2a-e; Extended Data Fig. 3a-k). However, later after damage, total and M2-like macrophages were fewer in Glud1 ΔMo vs. CTRL mice (Extended Data Fig. 3l,m), arguing that the faster resolution of inflammation is consequent to a more efficient muscle repair in Glud1 ΔMo mice.
Metabolic changes within a cell affect the biology of neighbouring cells 18 . In WT macrophages, glutamine oxidation was 73% lower in glutamine-reduced vs. glutamineenriched conditions (Fig. 2a). Compared to WT cells, glutamine oxidation in GLUD1 KO macrophages was lower in both culture conditions (Fig. 2a). However, total 2-oxoglutarate (2-OG) was comparable (Extended Data Fig. 4a), likely due to enhanced pyruvate carboxylase (PC)-dependent TCA-cycle anaplerosis (Fig. 2b), which compensates for the loss of glutamine-derived carbons 19 . Total energy charge, ATP production, ATP-linked oxygen consumption rate (OCR) were comparable in both genotypes (Extended Data Fig.  4b-d).
Though glutamine oxidation in GLUD1 KO macrophages was diminished, intracellular and extracellular glutamine production was higher under both glutamine-replete and, to a greater extent, glutamine-restricted conditions (Extended Data Fig. 4e,f). This was due to enhanced GS activity (Fig. 2c,d). At the protein level, GS was induced in WT macrophages under low glutamine but this induction was stronger in GLUD1 KO macrophages (Fig. 2e). Glutamine uptake and conversion into glutamate were similar in WT and GLUD1 KO macrophages (Extended Data Fig. 4g,h). GLUD1 converts glutamate into 2-OG but also 2-OG into glutamate, the latter used by GS to generate glutamine 12 . Consistent with this function, GLUD1 protein levels in WT macrophages were also upregulated under low glutamine (while undetectable in GLUD1 KO macrophages) (Fig. 2f). Under glutamine starvation, glucose utilization for glutamate production was enhanced in WT macrophages and even more in GLUD1 KO macrophages (Extended Data Fig. 4i,j). In absence of GLUD1, 2-OG into glutamate conversion was possibly taken-over by the increased activity of branched-chain amino acid aminotransferase (BCAT), utilizing branched-chain amino acids (leucine, isoleucine, and valine) as aminogroup donors, or glutamate-oxaloacetate transaminase (GOT), utilizing aspartate (Fig. 2g). However, only the silencing of cytosolic BCAT (BCAT1) restored glutamine production by GLUD1 KO macrophages back to the WT levels (Extended Data Fig. 4k-n). Consistently, the BCAT1 inhibitor gabapentin prevented SC expansion in CTX-treated Glud1 ΔMo mice (Extended Data Fig. 4o).
To assess the fate of macrophage-derived glutamine, we used a two-chamber co-culture of C2C12 myoblasts and macrophages in medium containing exclusively [ 13 C5, 15 N2]glutamine, i.e. glutamine labelled in the two nitrogen groups and in the five carbons. Uptake of [ 13 C5 15 N2]-glutamine by myoblasts did not change in all the conditions (Fig. 2h). However, compared to myoblasts alone, intracellular total glutamine (labelled and unlabelled) was lower in myoblasts cultured with WT macrophages but higher in myoblasts cultured with GLUD1 KO macrophages (Fig. 2h). In macrophages, total glutamine levels were always higher in GLUD1 KO macrophages (Fig. 2i) though a comparable uptake in all the conditions (Fig. 2i). Since in myoblasts, the contribution to the total glutamine pool of [ 13 C 0 15 N 0 ]-glutamine (derived from different sources than [ 13 C 5 15 N 2 ]-glutamine) was higher in co-culture with macrophages and peaked with GLUD1 KO macrophages (Fig. 2j), we argued that glutamine production by GLUD1 KO macrophages (Fig. 2j) outcompete glutamine consumption, increasing glutamine availability for myoblasts.
In vivo, interstitial glutamine levels in CTRL and Glud1 ΔMo muscles were similar at baseline and dropped in injured CTRL but not in Glud1 ΔMo muscles (Fig. 2k,l), whereas glutamate availability did not change (Extended Data Fig. 4p,q). GS depletion in macrophages worsened this post-injury glutamine shortage observed in control muscles, and also, disabled the preservation in glutamine levels seen in Glud1 ΔMo muscles (Fig. 2m). Similarly, macrophage-specific depletion of GS worsened SC proliferation and muscle healing after injury, and it impeded the faster SC activation and damage resolution seen in Glud1 ΔMo mice (Fig. 2n,o). Thus, macrophage GS is instrumental to replenish glutamine and promote SC activation in response to muscle damage (i.e. a glutamine-restricted condition).
Consistent with the above data, WT BMDMs placed in low glutamine showed reduced conversion of glutamate into 2-OG, a readout of oxidative GLUD1 activity (Extended Data Fig. 4r), whereas glutamine production involving the conversion of 2-OG into glutamate and glutamate into glutamine were both enhanced (Extended Data Fig. 4s,t). In muscleinfiltrating WT macrophages too, GS and oxidative GLUD1 activities, respectively, were gradually increasing and decreasing over time upon damage (Fig. 2p, upper panel), resulting in a slow increase of interstitial glutamine (Fig. 2p, Fig. 4u,v) which prevented the post-damage drop in interstitial glutamine (Fig. 2k).
We then linked macrophage-derived glutamine and myogenic potential. Myotube formation was promoted when differentiating C2C12 myoblasts were cultured in a glutamine-rich medium and reduced under glutamine restriction (Fig. 2q,r). Co-culture of C2C12 cells with WT macrophages impaired myogenic differentiation in glutamine-enriched conditions, phenocopying a condition of low glutamine. Instead, co-culture of C2C12 cells with GLUD1 KO macrophages resulted in larger myotubes, regardless of glutamine availability in the medium (Fig. 2q,r). Similar results were obtained with macrophage-conditioned media (Extended Data Fig. 5a,b). SLC1A5 knockdown (KD) in C2C12 cells impaired glutamine uptake (Extended Data Fig. 5c,d) and abrogated the advantage offered by GLUD1 KO macrophages on myoblast differentiation (Extended Data Fig. 5e,f). Conditioned media from GLUD1 KO macrophages enhanced the expression of both the proliferation marker Pcna and the differentiation marker Myogenin and activated mTOR pathway (an important player for SC proliferation/differentiation 20,21 ) in a glutamine-uptake-dependent manner 22 (Extended Data Fig. 5g,h). The mTOR inhibitor Torin2 abolished Pcna and Myogenin induction in C2C12 cells stimulated with conditioned media from GLUD1 KO macrophages (Extended Data Fig. 5g,h). Likewise, mTOR pathway was enhanced in SC isolated from CTX-injected Glud1 ΔMo vs. CTRL muscles (Fig. 2s).
In glutamine-replete conditions, SLC1A5-KD SC had impaired in vitro proliferation/ differentiation (Extended Data Fig. 5i-o). Therefore, to disrupt SLC1A5-mediated glutamine import specifically in SC in vivo, AAV8 particles containing the gRNA against Slc1a5 were injected intramuscularly in LSL-Cas9/PAX7:Cre-ERT mice, carrying a tamoxifen-inducible Cas9 in Pax7+ cells (Extended Data Fig. 6a-d). In this way SLC1A5 was selectively knocked-down by 60% in about all the SC (Extended Data Fig. 6e-h). Using bone marrow (BM) transplantation experiments, we confirmed that GLUD1 KO macrophages ameliorated muscle regeneration in mice with SC proficient in glutamine uptake (non-targeting control gRNA). Specifically, we administered EdU within 24h post-CTX and collected the injured TA muscles 6 days post-CTX. Compared to muscles in Glud1 WT BM (CTRL BM), those from Glud1 ΔMo BM mice treated with the non-targeting gRNA displayed a higher number of EdU+ terminally-differentiating myonuclei, increased MyoD+ myoblasts, higher number of Myogenin+ cells incorporated into the myofibers, as well as an increased area of regenerating myofibers (Fig. 2t-x). Conversely, when SC were perturbed in their capacity to import glutamine (Slc1a5-targeting gRNA), the superior myogenic potential of Glud1 ΔMo BM mice was abrogated but also severely affected in Glud1 WT BM (CTRL BM) mice ( Fig.  2t-x).
Mirroring muscle regeneration, tissue damage was milder in Glud1 ΔMo BM mice treated with the non-targeting gRNA, but worsened by the Slc1a5 gRNA in both CTRL and Glud1 ΔMo BM mice (Fig. 2y, Extended Data Fig. 7a-d). As the maximal SC expansion in a WT context occurs between day 2 and day 4 post-CTX 23 (Fig. 1r), we administered EdU until day 3. In this setting, the number of EdU+ myonuclei was much higher compared to a 24h EdU pulse and anyhow strongly reduced when SC were deleted for the glutamine transporter Slc1a5 (Extended Data Fig. 7e,f). Next to this approach, the SLC1A5 inhibitor gamma-L-Glutamyl-p-Nitroanilide (GPNA) impaired SC proliferation in both CTX-treated Glud1 ΔMo and WT mice ( Fig. 2z; Extended Data Fig. 5d). In sum, macrophage-derived glutamine uptake by SC plays a relevant role in the control of myogenic functions. Disrupting this cross-talk precludes the faster muscle repair in Glud1 ΔMo mice but also delays muscle regeneration in CTRL mice.
Regenerative functions are known to decline with aging 24 . Muscle weight index in 18 month-old CTRL mice was reduced when compared to young mice, but this drop was less severe in Glud1 ΔMo mice (Fig. 3a), as also indicated by the presence of larger myofibers (Fig. 3b,c). Interstitial glutamine followed the same trends (Fig. 3d). Fibrosis and macrophage infiltration were exacerbated in old vs. young CTRL mice, but to a lesser extent in Glud1 ΔMo mice ( Fig. 3e-g; Extended Data Fig. 8a), arguing that chronic inflammation inversely correlates with muscle health. Macrophage density in other tissues did not change (Extended Data Fig. 8b-i). Overall, muscle performance of 18 month-old Glud1 ΔMo mice was better than age-matched CTRL mice ( Fig. 3h-j). In line with this, Glud1 ΔMo muscles displayed increased numbers but decreased ratio of phospho-p38 positive vs. negative SC ( Fig. 3k-m), suggesting that SC self-renewal was better preserved in Glud1 ΔMo mice 25 .
We finally assessed the potential therapeutic effect of the GLUD1 inhibitor R16 2 26 . In response to CTX or ischemia, R162 treatment reduced muscle necrosis and inflammation ( Fig. 3n-p), and boosted SC proliferation and interstitial glutamine concentration (Fig. 3q,r). In 18 month-old mice as well, administration of R162 for a month increased muscle mass, SC numbers, and interstitial glutamine, and improved physical performance ( Fig. 3s-w). R162 did not affect body and organs' weights (Extended Data Fig. 8j-n).
This therapeutic benefit was selective for GLUD1 targeting. Unlike GLUD1 deletion (Extended Data Fig. 4g), macrophage-specific knockout of glutaminase (GLS), converting glutamine into glutamate, resulted in reduced glutamine uptake, glutamine oxidation, and 2-OG-to-succinate ratio but did not promote GS activity (Extended Data Fig. 9a-e). Six days post-CTX, interstitial glutamine levels, muscle damage, and macrophage infiltration were comparable in CTRL and Gls ΔMo mice, but macrophages were more M1-like in Gls ΔMo mice (Extended Data Fig. 9f-m), consistent with in vitro data (Extended Data Fig. 9n-q) and previous findings 27 .
Muscle tissue is a major site for glutamine synthesis in the body 9,10,11 . We show here that muscle damage and aging restrain glutamine availability. Muscle-infiltrating macrophages sense this shortage and tilt down glutamine oxidation in favor of glutamine production. Macrophage-released glutamine is uptaken by SC promoting their proliferation and differentiation through mTOR activation. Unlike WT macrophages, GLUD1 KO macrophages are pre-adapted to glutamine starvation, which results in improved regeneration (Extended Data Fig. 9r). Thus, a metabolic rewiring in macrophages reestablishes muscle homeostasis in response to damage.
The risk of vascular occlusion and skeletal muscle impairment is increased in case of diabetes, hypercholesterolemia, and obesity, which are recurrent in today's society 28 .
Similarly, sarcopenic patients are arising together with the population age 29 . However, little can be done to improve these conditions 28,30 . Our data suggest a pharmacologic approach to treat damage and age-related skeletal muscle decline.

Cardiotoxin muscle injury
Mice were anaesthetized with isoflurane and 50 μl of 10 μM CTX was injected in the tibialis anterior (TA) muscle 14 . Control mice (denoted as baseline) were subjected to PBS injection.
For SC isolation, mice were injected with 50 μl of 10 μM CTX in TA and 100 μl of 10 μM CTX in gastrocnemius muscles. Muscles were harvested for analysis at different time points post-injury. In vivo GLUD1 inhibition was achieved by bi-daily gavage of R162 (Focus Biomolecules) at 0.6 mg/mouse. Mice were pretreated 1 day before CTX injection. Afterwards, mice continued to receive bi-daily treatment until their sacrifice. To inhibit SLC1A5, mice were treated by oral gavage 3 times per day with a solution (200 μl/mouse) containing 500 mM GPNA (Sigma-Aldrich). CTX was injected 1 h after the first gavage, and mice were sacrificed 24 h afterwards. In vivo BCAT1 inhibition was achieved by bidaily i.p. injection of gabapentin (Sigma-Aldrich) at 2 mg/mouse. Mice were pretreated once before CTX injection, and mice were sacrificed 24 h afterwards.

Hindlimb ischemia
To induce acute hindlimb ischemia and greatly prevent flow redirection into the collateral circulation, which leads to severe muscle necrosis, unilateral or bilateral ligations of the high femoral artery were performed without damaging the nervus femoralis as previously described 15 . Control mice (denoted as baseline) were subjected to a sham operation that did not involve the ligation of the femoral artery. Functional perfusion measurements were performed using a Lisca PIM II camera (Gambro) 15 . GLUD1 inhibition was achieved by bidaily gavage of R162 (Focus Biomolecules) at 0.6 mg/mouse. Mice were pretreated 1 day before femoral artery ligation. Afterwards, mice continued to receive bi-daily treatment until their sacrifice.

Wheel running test
Physical activity was assessed with voluntary wheel running test. Mice were individually housed in cages equipped with 12 cm diameter wheels for rodents, wheels were connected with cycle computers (SunDing SD-568AE). After 1 week of acclimatization, physical activity (i.e. duration, running speed, and distance) was recorded daily.

Rotarod test
Whole body mobility and coordination was assessed by rotarod performance. Following a 5 min acclimatization in the test room, mice were placed on the rod (Biological research apparatus), which was rotating at an initial speed of 4 rpm. The speed was increased gradually from 4 rpm to 40 rpm within 5 min and latency to fall on to a soft pad was recorded. The test was repeated twice more, with 15 min between tests. After 3 days training, latency to fall was calculated over 3 trials.

Grip test
Muscle strength was measured by pulling backwards the mice with a continuous movement when the mice were hold firmly to the grip of strength meter (Chatillon, DFE II Digital Force Gauge). The test was repeated twice more, with 15 min between tests. Results was calculated over the 3 trials.

TTC staining
TA muscles were collected 6 days after CTX injection. After scarification muscles were washed in PBS and cut in transversal sections. Sections were incubated with a buffered (pH 7.4) 1.5% 2,3,5-triphenyl-tetrazolium chloride (TTC) solution at a temperature of 37°C for 40 min. Pictures were taken with NIKON camera. The quantification was performed with Image J software. Images were converted in a grey scale and it was calculated the mean grey intensity of each section.

Interstitial fluid preparation
Intact TA muscles for the CTX model, or crural muscles (gastrocnemius and soleus muscles) for the ischemia model, were placed into test tubes with perforated bottom. 20 μL of 0.9% NaCl solution (pH 7.4) was added to the sample. Interstitial fluid was collected by centrifugation (110 g, 10 min, 4°C). Protein within theinterstitial fluid was precipitated using -20°C cold methanol/water-mix (5:3) and centrifuged (20000 x g, 5 min, 4°C). The supernatant was dried using a vacuum centrifuge and derivatized for mass spectrometry analysis.

Bone marrow transplantation
5-6 weeks old recipient mice were irradiated with 9.2 Gy. Subsequently, 10x10 s bone marrow cells from the appropriate genotype were injected intravenously via the tail vein.
Muscle injury experiments were initiated 5 weeks after bone marrow reconstitution. Red and white blood cell count was determined using a hemocytometer on peripheral blood collected in heparin with capillary pipettes by retro-orbital bleeding.

In vitro and in vivo targeting of the Slcla5 locus
To generate in vitro a stable C2C12 mouse myoblast cell line and primary SC, deficient for SLC1A5, lentiCRISPRv2 vectors expressing the Cas9 along with a gRNA targeting the Slcla5 locus (AATCCCTATCGATTCCTGTG) or a non-taregting control gRNA (GAACAGTCGCGTTTGCGACT), were used. 2xl0 4 C2C12 cells/well were seeded in 12well plates and transduced with purified lentiviral vectors in the presence of 8 Mg/ml polybrene for 48 h. After adding virus, the plate was centrifuged for 30 min at 800x g, 25-32°C. Transduced cells were selected with puromycin (4 μg/ml) during a period of 5 to 7 days. The lentiCRISPRv2 was a gift from Dr. Feng Zhang (Addgene plasmid # 52961) 36 . The gRNAs were cloned as described previously 36 . For an optimal transduction of SCs cells in vivo and specifc targeting of the Slcla5 locus exclusively in SCs, the gRNAs described above were cloned in the AAV_Guide vector (see also Extended Data Fig. 6a). AAV8 production and titration was outsourced to Vigene Biosciences (Rockville, MD 208050, USA). Two-day-old (P2) neonatal LSL-Cas9 x PAX7:Cre-ERT transgenic mice were then administered locally into the TA muscles with 6.7 X 10 10 vector genomes (vg) per injection (5 μl/leg). Bone marrow cells from CTRL and Glud1 ΔMo mice were transplanted at the age of five weeks. Five weeksafter bone marrow reconstitution, tamoxifen was given by daily oral gavage for five days before and three days after CTX induced injury. 180 μg EdU was given by i.p. injection 24 h after CTX injection. 3 hours after CTX injection mice also received a second AAV injection into the TA muscles (4 X 10 11 vg per injection, see shematic in Extended Data Fig. 6b).

SC isolation and culture conditions
Hindlimb muscles were dissected and minced in small fragments by scissors and digested in 0.1% collagenase/0.25% trypsin solution at 37°C with gentle shaking for 20 min as previously described 37 . Supernatants were collected in fetal bovine serum (FBS) at 37°C and passed through 40 μm filters. The digestion step was repeted 3 times until the complete dissociation of samples. The fraction of freshly isolated SC was enriched by using the SC isolation kit (Miltenyi Biotec, 130-104-268). SC were collected on glass slides by cytospin for immunofluorescence analyses, or snap frozen for protein extraction. The fraction of non-SC (Miltenyi Biotec, 130-104-268) was used as control where indicated.
EdU-based SC proliferation assays were performed by seeding in 6 well plates 3.0 x 10 4 cells/well in GM containing 10 μM of 5-ethylnyl-2'-deoxyuridine (EdU) (Thermo Fisher, C10337) for 24 h prior the fixation. Click-iT ® reaction and staining were performed according to the manufacturer's instructions.
Differentiation assays were performed by seeding 1.0 x 10 4 /cm 2 SC in GM. After 24 h, GM was replaced with DM for 5 days. Myotubes-derived SC were fixed in 4% paraformaldehyde and stained for myosin heavy chain. Fusion index (percentage of nuclei within myotubes: myotube=nuclei>2) and myotube size (mean of number of nuclei into myotubes) were analyzed.

Single fiber isolation and immunostaining
Single myofibers were isolated from extensor digitorum longus (EDL) muscles of aged mice as previously described 37 . Briefly, intact muscles were dissected from tendon to tendon and digested with a solution containing 0.2% collagenase type I (Sigma) in DMEM (Dulbecco's modified Eagle's medium; high glucose, L-glutamine with 110 mg/ml sodium pyruvate) at 37°C for 3h. Afterward, individual fibers were separated from each others by pipetting under a dissecting microscope, and then washed in DMEM.

Cell lines
C2C12 murine myoblast cells were obtained from the American Type Culture Collection (ATCC). C2C12 authentication was confirmed by testing their myogenic differentiation capacity. Specifically, RT-qPCR and immunofluorescence analyses of the expression of specific myogenic markers (e.g. myogenin and myosin heavy chain) confirmed their myogenic origin. Cells were regularly tested for mycoplasma via PCR.

Glutamine-enriched and glutamine-reduced media preparation
When specified, the glutamine (Q)-enriched medium (4mM glutamine) was obtained by supplementing M199 or DMEM with 10% FBS. A glutamine (Q)-reduced medium (0.03 mM glutamine) was obtained by supplementing M199 or DMEM (with no glutamine) with 10% FBS, dialyzed for glutamine. Dyalized FBS was obtained by slide A lyzer dialysis cassette (Thermo Scientific 66130).
In the proliferation assay, 6 x 10 4 C2C12 cells/well were seeded in 6-well plates in GM. After 24 h, regular GM was replaced with a Q-reduced GM that was previously conditioned by WT or GLUD1 BMDMs.
In the differentiation assay, 6 x 10 4 C2C12 cells/well were seeded in a 6-well plates in GM. After 24 h, GM was replaced with Q-enriched or Q-reduced differentiation medium (DM), that is the same medium as described above for SC. At day 2 of myoblast differentiation, 1.5 x 10 5 BMDMs or BMDM-conditioned DM were added to the differentiating myotubes for additional 4 days. Afterwards, samples were collected for RNA or fixed in 4% formaldehyde for 10 min at RT and permeabilized in PBS with 1% BSA, 0.2% TritonX for 30 min and blocked in 10% Donkey serum lh at RT. Finally, samples were incubated overnight at 4°C with mouse anti-MF20 (DSHB, 2μg/ml) and subsequently incubated with AlexaFluor 568conjugateddonkey anti-mouse (Invitrogen 1:1000). When indicated, mTOR inhibition was achieved by adding 500nM Torin2 (Selleck Chemicals) to the medium.

Glutamine oxidation
Each M199 media (Q-enriched or Q-reduced) was supplemented with 0.5 μCi/ml [U-14 C]glutamine. After incubating 1 x 10 6 BMDMs in a 12-well plate for 6h, 250 μl of 2 M perchloric acid was added to each well to stop cellular metabolism. Each well was immediately covered with a 1 x hyamine hydroxide-saturated Whatman paper. Overnight absorption of 14 CC>2 released during the oxidation of glutamine into the paper was performed at RT and radioactivity in the paper was determined by liquid scintillation counting.
Glutamine uptake 2 x 10 6 BMDMs were seeded in a 6-well plate and cultured in M199 medium (Gibco) and 10% FBS, supplemented with 0.5 μCi/ml [U-14 C]-glutamine for 30 min at 37°C. Cells were lysed in IN NaOH and the radioactivity was measured by liquid scintillation counting.

Metabolites quantification by LC-MS/MS
For mass spectrometry analysis of glutamate and glutamine, 2 x 10 6 cell pellets were washed twice in PBS and extracted in 500 μl of 80% methanol. Upon extraction, samples were centrifuged at 20000 x g for 15 min and the supernatant was dried using a vacuum centrifuge. 25 μl of a 2% methoxyamine hydrochloride solution were added to the dried pellet and the tubes were then placed at 37°C for 90 min. Then 75 μl of N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide with 1% N-tert-Butyldimethyl-chlorosilane (Sigma-Aldrich, Bornem, Belgium) was added and the reaction was carried out for 30 min at 60°C. Reaction mixtures were then centrifuged for 15 min at 20000 g at 4°C in order to remove insolubilities, the supernatant was transferred to a glass vial with conical insert (Agilent). GC-MS analyses were performed using an Agilent 7890A GC equipped with a HP-5 ms 5% Phenyl Methyl Silox (30 m -0.25 mm i.d. -0.25 μm; Agilent Technologies, Santa Clara, California, USA) capillary column, interfaced with a triple quadruple tandem mass spectrometer (Agilent 7000B, Agilent Technologies) operating under ionization by electron impact at 70 eV. The injection port, interface and ion source temperatures were kept at 230°C. Temperature of the quadrupoles was maintained at 150°C. The injection volume was 1 μl, and samples were injected at 1:25 split ratio. Helium flow was kept constant at 1 ml/min. The GC oven temperature was held at 60°C for 3 min, increased to 300°C at 9°C/ min, and kept for 2 min. The mass spectrometer operated in SIM mode, glutamine and glutamate were determined from the m/z 341.2 and 342.2 respectively. For quantifications, the MassHunter Workstation Software B.06.00 SP01 (Agilent Technologies) was used.

In vivo GS activity and glutamate to 2-OG conversion
GS activity and glutamate to 2-OG conversion in muscle-infiltrating macrophages, sorted 1 and 3 days after CTX injection, were measured respectively with the glutamine synthetase microplate assay kit (ABIN2704091, Cohesion Biosciences) and the glutamate dehydrogenase activity assay kit (MAK099-1KT, Sigma-Aldrich).

TPA model of acute skin inflammation
Phorbol ester TPA was used to induce acute skin inflammation as described before 38 . Briefly, TPA (2.5 μg in acetone, 20 μl total volume/site) was topically applied on the ear skin of anaesthetized mice. As vehicle control, the ear was painted with acetone alone. After the indicated time points, tissue was harvested for further analysis.

FACS analysis of muscle macrophages
TA muscles were dissected, dissociated mechanically, digested using 800U/ml collagenase II (10 ml per sample) for 1 h at 37°C, centrifuged and resuspended with lOOOU/ml collagenase II (1 ml per sample) and llU/ml Dispase (1 ml per sample) solution and incubated for 30 min at 37°C. The digested tissue was filtered using a 40 μm pore sized mesh and cells were centrifuged 5 min at 500 g. Cells were resuspended in

Histology and immunostainings
7 μm-thick cryosections were obtained by using a Leica cryostat from frozen muscles collected in optimal cutting temperature compound (OCT) and fixed in 4% formaldehyde for 10 min at RT. Alternatively, TA and crural muscle were fixed in 2% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned at 7 μm thickness. Necrotic muscle area was detected by H&E staining as the area which includes necrotic myocytes, inflammatory cells and interstitial cells. After deparaffinization and rehydration, muscle sections were permeabilized by a solution containing 1% BSA, 0.2% Triton-X in PBS 30 min at RT, blocked with 10% Donkey serum (Sigma) 1 h at RT and treated with target retrieval solution at pH 6.1 (Dako, S1699) at 92°C for 20 min. To reduce the immune background sections were blocked with 10% donkey serum in PBS, lh at RT, followed by blocking with FAB fragment anti-mouse IgG (Jackson Immunoresearch were performed according to the manufacturer's instructions. Immunofluorescence of SC cytospinned on glass slides was performed by fixation in 4% formaldehyde for 10 min at RT, followed by incubation with 0.5% Triton-X in PBS for 20 min at RT and blocking with with 10% donkey serum for 1h at RT. Samples were then probed with mouse anti-Pax7 (DSHB, 1:20) alone or incombination with rabbit anti-SLC1A5 (Alomone Labs, 1:50) for 2h at RT followed by incubation with mouse Alexa 568 (Invitrogen, 1:10000), or a combination of mouse Alexa 488 and rabbit 568 conjugated secondary antibodies (Invitrogen, 1:1000), respectively. In vivo cell proliferation and differentiation was detected by DNA incorporation of thymidine analogue EdU (ThermoFisher, Click-iT C10337) in combination with Dystrophin staining on muscle cryosections. Oxidative damage was detected by dihydroethidium (DHE) (Life Technology). Samples were incubated with 10 ^M DHE at 37°C for 30 min. Apoptosis was detected by TUNEL assay kit (Sigma-Aldrich) according to the manufacturer's instructions. Nuclei were counterstained with Hoechst-33342 (Invitrogen, 1:1000). Whenever sections were stained in fluorescence, ProLong Gold mounting medium with or without DAPI (Invitrogen) was used.

Imaging and Morphometry
Images were acquired by an Olympus BX41 microscope equipped with the CellSense Dimension imaging software. CellSense Dimension software was used for the morphometric analyses of cell cultures and muscle tissue. Myotubes diameter was measured as the average from three independent measurements per myotube.

Bone marrow-derived macrophages (BMDMs)
Macrophages were derived from bone marrow precursors as described before 38 . Briefly, bone marrow cells (1.6 x 10 6 cells/ml) were cultured in a volume of 6 ml in a 10 cm Petri dish in DMEM supplemented with 20% FBS and 30% L929 conditioned medium as a source of M-CSF. After 3 days of culture, an additional 3 ml of differentiation medium was added. At day 7, macrophages were harvested with ice cold Ca2 + and Mg2 + -free PBS. The cells obtained were uniformly macrophages as assessed by FACS, using the pan-macrophage marker F4/80. When indicated, GS inhibition in cultured BMDMs was achieved by adding 1 mM L-methionine-SR-sulfoximine (MSO; Sigma) to the medium for 48 h. Silencing of Bcatl, Bcat2, Gotl, Got2 and Alt in BMDMs was achieved by electroporation with specific siRNAs (IDT). Briefly, 5.6 xlO 6 BMDMs were resuspended in 750 μl of Opti-MEM and were electroporated (250V, 950 μF, ∞ Ω) with 120 pmol of total siRNA IDT. Control BMDMs were electroporated with scrambled siRNA sequences.

BMDM migration assay
Migration of BMDMs was assessed by using a 8-μm-pore Transwell permeable plate (Corning Life Science). The bottom chambers contained DMEM with specific chemoattractants or controls (specified in each Figure), BMDMs were harvested and then seeded in the upper chamber (2.5 x 10 5 cells in 200 μl of DMEM at 2% HS). After 4 h incubation, migrated cells were fixed with 4% paraformaldehyde, stained with 5mg/ml crystal violet/20% methanol and counted under the microscope.

Macrophage phagocytosis
BMDMs were treated with 50 ng/ml of IL-4 in DMEM complete, non-treated condition was used as control. 6 h after treatment, cells were harvested and incubated 40 min at 37°C with latex beads (1:5000) (Polysciences Ref. # 17152-10), negative controls were incubated 40 min at 4°C. After incubation, cells were washed with 2ml MACS buffer, and read by FACS in the FITC channel.

Endothelial sprouts
BMDMs were treated with 10 ng of IL-4 in DMEM complete, non-treated condition was used as control. 24 h after treatment, macrophages were harvested and incubated overnight with HUVECs (1400 BMDMs and 1400 HUVECs) in hanging drops of 25 μL in EGM-2 medium containing methylcellulose (methylcellulose 4000 cP, Sigma-Aldrich, Bornem, Belgium) to form spheroids. Then, spheroids were harvested and embedded in collagen type I gel in a 24-well plate and cultured for 20 h to induce sprouting. Spheroids were fixed with 4% PFA and images were captured with a LEICA DM1600B inverted light microscope. The total sprout length per spheroid (cumulative length of primary sprouts and branches) was done manually using the Image J software.

RNA sequencing
RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 4 ng of total RNA was used as input for the SMART-Seq v4 Ultra Low Input RNA protocol (version "091817") from Takara Bio USA, Inc. Subsequently, 5 ng of purified cDNA was sheared to 300bp using the Covaris M220. From the sheared material, sequencing libraries were prepared with the NEBNext Ultra DNA Library Prep Kit for Illumina (version 6.0 -2/18), according to the manufacturer's protocol including a size selection to 250bp insert size. Sequence-libraries of each sample were finally equimolarly pooled and sequenced on 1 NextSeq500 v2 flow-cell at 1x75 bp (76-6-0-0).

Statistics
Data entry and all analyses were performed in a blinded fashion. All statistical analyses were performed using GraphPad Prism software on mean values calculated from the averages of technical replicates. Statistical significance was calculated by two-tailed unpaired t-test on two experimental conditions or two-way ANOVA when repeated measures were compared, with P<0.05 considered statistically significant. A representative (everywhere except for n) or a pool (n) of at least two independent experiments is shown. Unpaired two-tailed t-test was everywhere applied; ns, not significant (P>0.05). Scale bars: 50 μm (k). Graphs show mean ± SEM.   (n=3). u,v, Evaluation of the conversion of GLUD1 activity (u), and glutamine synthetase (GS) activity (v), in muscle-infiltrating macrophages, sorted 1 day post-CTX. One unit for the conversion of glutamate to 2-OG is the amount of enzyme that will generate 1 μmole of NADH per minute at pH 7.6 at 37°C. One unit of GS activity is defined as the enzyme producing 1 nmole of gamma-glutamyl hydroxamic acid per minute (CTRL n=4; Glud1 ΔMo n=3). The control condition (CTRL) in u,v is the same one displayed in Fig. 2p (n=3). f, Fold change in glutamine to glutamate ratio in the interstitial fluid of TA muscle 1 day post-CTX, relative to PBS injected CTRL muscle (n= 6). g,h, Quantification of necrotic (right side of the graph) and regenerating (left side of the graph) areas on H&E-stained sections from TA muscles 6 days post-CTX (n= 6) (g) and representative images (h). i,j, Quantification (i) and representative images (j) of TUNEL + cells in TA muscle 6 days post-CTX (n=6). k-m, Representative images (k) and quantification of F4/80 + area (l), CD206 -F4/80 + cells (M1) to CD206 + F4/80 + cells (M2) ratio (m) in TA muscles 6 days post-CTX (n=6). n-q, RT-qPCR of Tnfa (n), Cxcl9 (o), Mrc1 (p) and Retnla (q) in BMDMs isolated from CTRL and Gls ΔMo mice. BMDMs were treated with LPS or PBS (unstimulated) prior to the assay (n=3). r, Scheme illustrating the physiological role of Glud1 in macrophages in response to muscle damage. During muscle disruption, ischemia or aging, interstitial glutamine drops likely because of the loss in myofibers (a major glutamine source) and poor blood supply. Infiltrating macrophages respond to glutamine starvation by reducing their oxidative GLUD1 activity in favour of GS activity. Macrophage-derived glutamine is released and progressively fills the muscle interstitium, where it is uptaken by SC promoting their proliferation and differentiation into new fibers, two processes that are favoured by glutamine-dependent mTOR activation. Towards the end of this regenerative process, the newly generated fibers will undertake glutamine production while inflammation will be progressively resolved. GLUD1-deficient macrophages are metabolically pre-adapted towards glutamine synthesis and release, thus preventing this glutamine drop. It follows that, in case of muscle damage, macrophage-specific knockout of Glud1 or pharmacologic GLUD1 blockade strengthens SC activation, ultimately leading to therapeutic muscle regeneration. All experiments show representative values of at least 2 independent experiments. Unpaired two-tailed t-test was everywhere applied; ns, not significant (P>0.05). Scale bars: 20 μm (h); 10 μm (j,k). Graphs show mean ± SEM.

Europe PMC Funders Author Manuscripts
Europe PMC Funders Author Manuscripts