Nicotinamide mononucleotide ameliorates high glucose/high fat-induced cardiomyocyte metabolic dysfunction through SIRT1-mediated CPT1A stabilization

To investigate the mechanism of nicotinamide mononucleotide (NMN) in ameliorating high glucose/high fat (HG/HF)-induced metabolic dysfunction in diabetic cardiomyopathy (DCM) through SIRT1-mediated CPT1A stabilization. DCM cellular model was established using H9c2 cell. After screening optimal NMN concentration via cell counting kit-8 (CCK-8) assay and Western blot, cellular viability, apoptosis, total reactive oxygen species (ROS), mitochondrial function, ATP, and β-hydroxybutyrate (β-OHB) content were measured. The molecular interplay among NMN-SIRT1-CPT1A was further elucidated through co-immunoprecipitation (Co-IP), cycloheximide (CHX) chase assay, MG132 rescue, and CPT1A K675R mutation. HG/HF reduced H9c2 cells viability by 26.66% and SIRT1 protein expression by 79.30%, both of which were restored by 100 µM NMN. In vitro, NMN enhanced cell viability, suppressed apoptosis and total ROS, stabilized mitochondrial function, and increased ATP and β-OHB content, these protective effects were attenuated by SIRT1 knockdown. Western blot analysis demonstrated NMN upregulated CPT1A and CD36 expression by activating SIRT1. Co-IP revealed that HG/HF markedly elevated the acetylation and ubiquitination of CPT1A, both of which were weakened by NMN treatment. Moreover, SIRT1 directly interacted with CPT1A and deacetylated CPT1A via the proteasomal pathway, thereby blocking its ubiquitination. Additionally, the K675R point mutation further confirmed Lys675 as the specific deacetylation target of SIRT1 on CPT1A. NMN activates SIRT1 to deacetylate CPT1A at Lys675, inhibiting its degradation and enhancing mitochondrial ATP and β-OHB generation, thereby mitigating HG/HF-induced injury. These findings provide SIRT1-mediated CPT1A stabilization as a potential therapeutic target for DCM.