Extended Data Figure 4. GLUD1 loss in macrophages enhances GS-mediated glutamine release.

a, Quantification (by GC-MS) of intracellular 2-oxoglutarate content in BMDMs cultured in Q-enriched or Q-reduced media (n=3).

b,c, LC-MS measurement of total cellular energy charge ([ATP + 1/2ADP]/[ATP + ADP + AMP]) (b) and ATP content (c) in BMDMs (n=3).

d, Oxygen consumption rate (OCR) in BMDMs (n=5).

e-f, Quantification of intracellular (e) and extracellular (f) glutamine content in BMDMs cultured in Q-enriched or Q-reduced media (n=3).

g, [U-¹⁴C]-glutamine uptake in BMDMs cultured in Q-enriched (n=4) or Q-reduced (WT n=4; Glud1^ΔMo n=3) media.

h, Evaluation of [U-¹³C]-glutamine-derived carbon incorporation into glutamate in BMDMs (n=3).

i-j, Evaluation of [U-¹³C]-glucose-derived carbon incorporation levels into 2-oxoglutarate (i) and glutamate (j) in BMDMs (n=3).

k-l, Quantification of intracellular (k) and extracellular (l) glutamine content in BMDMs upon silencing of BCAT1 or BCAT2 (n=3).

m-n, Quantification of intracellular (m) and extracellular (n) glutamine content in BMDMs upon silencing of GOT1 or GOT2 (n=3).

o, Quantification of SC on TA muscles 1 day post-CTX injury, stained for PHH3 and Pax7. CTRL and Glud1^ΔMo mice were treated 2 times per day with the BCAT1 inhibitor Gabapentin, or vehicle as control (n=6).

p, Fold change in glutamate to leucine ratio in the interstitial fluid of TA muscles 1day post-CTX, relative to PBS-injected CTRL muscle (PBS n=6; CTX n=9).

q, Fold change in glutamate to leucine ratio in the interstitial fluid of crural muscles 3 days post-ligation, relative to CTRL baseline muscle (Baseline n=7,8 CTRL, Glud1^ΔMo, respectively; ligated n=11,12 CTRL, Glud1^ΔMo, respectively).

r, Evaluation of the conversion of glutamate to 2-OG by analyzing [U-¹³C]-glutamine (Q-enriched condition) or [U-¹³C]-glutamate (Q-reduced condition) incorporation into 2-OG in WT BMDMs (n=3).

s, Evaluation of the conversion of 2-OG to glutamate by analyzing ¹⁵NH₄ ⁺ incorporation into glutamate in WT BMDMs (n=3).

t, Evaluation of glutamine synthetase (GS) activity by analyzing ¹⁵NH₄ ⁺ incorporation into glutamine in BMDMs (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 at day 1.

All experiments (except for o) show representative values of at least 2 independent experiments, o shows values from one single experiment. Unpaired two-tailed t-test was everywhere applied; ns, not significant (P>0.05); a.u., arbitrary unit. Graphs show mean ± SEM.