**Background:** Skeletal muscle growth and function are influenced by the gut–muscle axis, where microbial metabolites play key roles. While some metabolites (e.g., short-chain fatty acids) promote muscle health, others like d-type metabolites (e.g., d-amino acids, d-lactic acid) have been implicated in disease. d-Malate, produced by certain bacteria, has unknown physiological functions. This study investigates the origin, age-related changes, and effects of d-malate on skeletal muscle and angiogenesis, and explores underlying mechanisms.
**Methods:** The study used C57BL/6 male mice. To determine d-malate origin, mice were treated with antibiotics (1 mg/mL metronidazole, penicillin, streptomycin) for 1 week, and d-malate content in intestinal segments was measured. In vitro intestinal content cultures with 500 μM l-malate assessed microbial conversion. d-Malate levels in serum and colon content were compared in young (6 weeks), mature (16 weeks), and aging (24 months) mice. For functional studies, 4-week-old mice were fed chow with 2% d-malate or l-malate for up to 10 weeks. Outcomes included body weight, food intake, body composition (nuclear magnetic resonance), energy metabolism (Promethion system), muscle strength (weight test), muscle mass (gastrocnemius, soleus, extensor digitorum longus, tibialis anterior), muscle fiber cross-sectional area (laminin staining), fiber type (MyhC I/IIb immunofluorescence), and capillary/arteriole density (CD31, α-SMA staining). Protein expression (VEGFR2, VEGFB, HIF1α, HIF2α, VEGFA, VEGFR1, ubiquitin, P-AKT, P-FoxO3, LC3) and mRNA (mitochondrial, mitophagy, cell cycle genes) were analyzed by western blot and qPCR. In vitro, C2C12 myoblasts, primary vascular smooth muscle cells (VSMCs), and pulmonary vascular endothelial cells (PVECs) were treated with d-malate (20–500 μM). Cell proliferation (CCK-8, EdU), cell cycle (flow cytometry), tube formation, migration (scratch test), and d-malate uptake were assessed. Transcriptomics (RNA-seq) on PVECs identified differentially expressed genes. Acetyl-CoA content, total protein acetylation, and Cyclin A acetylation (immunoprecipitation) were measured. The p300 inhibitor C646 was used to test acetylation dependence. A VEGFB-induced angiogenesis mouse model (250 ng VEGFB protein injected every other day for 2 weeks) was used to verify the role of angiogenesis in d-malate-induced muscle loss.
**Key Results:** Antibiotic treatment significantly reduced d-malate in duodenum, ileum, cecum, and colon (Fig. 1A). In vitro, microbiota increased d-malate from l-malate (Fig. 1B). Aging mice (24 months) had significantly higher d-malate in serum and colon content compared to young and mature mice (Fig. 1C,D). Dietary d-malate (2%) increased serum d-malate and decreased body weight gain and muscle mass without affecting food intake or fat mass (Fig. 2A–D). d-Malate reduced oxygen consumption and energy expenditure but not respiratory quotient or activity (Fig. 2E–L). Muscle strength was reduced (Fig. 3A). Gastrocnemius, soleus, and extensor digitorum longus weights decreased (Fig. 3B–F). Muscle fiber cross-sectional area decreased in gastrocnemius, soleus, and EDL (Fig. 3G,H, EV1A–F). Type I fiber proportion decreased, type IIb increased in gastrocnemius (Fig. 3I–L). Ubiquitin protein increased, mitophagy genes (Bnip3, DRP1) mRNA decreased (Fig. 3M–O). No changes in serum cortisol, P-AKT, P-FoxO3, LC3, or mitochondrial function genes (Fig. EV1G–M, 3O). d-Malate did not affect C2C12 proliferation, differentiation, or P-mTOR/MyHC expression (Fig. EV2). Capillary and arteriole numbers in gastrocnemius decreased (Fig. 4A–D). VEGFR2 and VEGFB protein decreased; HIF1α, HIF2α, VEGFA, VEGFR1 unchanged (Fig. 4E–L). In VSMCs, d-malate inhibited proliferation but not cell cycle or vasomotor gene expression (Fig. 5A–C, EV3A–E). In PVECs, d-malate inhibited proliferation, reduced G1 phase, increased G2 phase, and decreased Cyclin A and Cyclin B mRNA (Fig. 5D–I, EV3F). Tube formation and migration were inhibited (Fig. 5J–M). d-Malate uptake was higher in PVECs than C2C12 (Fig. 5N). VEGFB injection rescued d-malate-induced reductions in lean mass, gastrocnemius mass, and muscle fiber area (Fig. 6A–F). d-Malate increased ROS but ROS scavenger NAC did not rescue tube formation (Fig. EV4A–C). Transcriptomics showed altered expression of acetyl-CoA-related genes (PDE4D, SUZ12, BRD7, ACAT2, LMNA, RUVBL2) (Fig. 7A–C). Acetyl-CoA content and total protein acetylation increased (Fig. 7D–F). Cyclin A acetylation increased (Fig. 7G,H). p300 inhibitor C646 blocked d-malate's effect on tube formation (Fig. 7I,J).
**Clinical Implications:** This study identifies a novel gut–muscle axis mechanism where microbiota-derived d-malate, elevated during aging, inhibits skeletal muscle growth and angiogenesis by promoting Cyclin A acetylation in endothelial cells, leading to G2 arrest. The findings suggest that targeting intestinal d-malate production or its downstream acetylation pathway could be a therapeutic strategy for age-related sarcopenia and impaired vascularization. The rescue of muscle loss by VEGFB highlights angiogenesis as a critical mediator. These results provide an experimental basis for developing interventions (e.g., modulating gut microbiota, inhibiting d-malate production, or blocking Cyclin A acetylation) to preserve muscle mass and function in aging populations.