Lactate as an Exercise Mimetic: Mitigating Disuse Atrophy and Improving Muscle Endurance in Aging SAMP8 Mice
Description
Lactate, historically considered a metabolic byproduct, has emerged as a key regulator of muscle physiology and metabolism. This study explores its potential as an exercise mimetic to counteract disuse muscle atrophy (DMA) in aging skeletal muscle using a hindlimb suspension model in senescence-accelerated prone 8 (SAMP8) mice. The mice were divided into four groups: Control, lactate-treated control, hindlimb suspension, and hindlimb suspension with lactate intervention. Lactate administration significantly preserved gastrocnemius muscle mass, restored muscle strength, and attenuated oxidative muscle fiber atrophy. Electrophoretic and histological analyses revealed an increase in MyHC I expression, indicating a protective effect on oxidative muscle fibers. Functional assessments showed that lactate improved muscle endurance and contractile force, while metabolomic profiling identified significant changes in energy metabolism, amino acid metabolism, and protein synthesis pathways. Specifically, lactate intervention improved impaired branched-chain amino acid metabolism, suggesting improved protein synthesis and recovery. In addition, lactate enhanced Cori cycle activity, upregulated hepatic lactate transporters, and increased lactate dehydrogenase B enzymatic activity, facilitating efficient lactate metabolism and gluconeogenesis. These results provide new insights into the role of lactate as a metabolic regulator and highlight its potential as a therapeutic intervention to combat exercise-induced muscle wasting and preserve muscle function in aging and immobilized individuals.
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1. Preparation of gastrocnemius muscle samples for Western blot analysis For protein expression analysis, gastrocnemius muscle tissue was homogenized in RIPA lysis buffer supplemented with protease and phosphatase inhibitors to prevent enzymatic degradation. Cold homogenization was performed to preserve protein integrity, followed by ultrasonic disruption to ensure complete cell lysis and efficient protein extraction. The homogenized lysates were centrifuged at 12,000g for 10 min at 4°C to allow separation of soluble proteins in the supernatant from cellular debris. The clear supernatant containing extracted proteins was carefully transferred to new tubes for further analysis. Protein concentrations were determined using the BCA Protein Assay Kit to ensure standardization across samples. SDS-PAGE loading buffer was added to achieve consistent protein concentrations to facilitate reliable comparative analysis. For electrophoresis, protein samples were denatured at 95°C for 10 minutes, effectively disrupting tertiary and quaternary structures to yield linearized proteins suitable for gel separation. Samples were either processed immediately or stored at -80°C for future use. 2.Protein expression detection and analysis Protein samples were separated by SDS-PAGE using gradient gels (8%-15%), which allows efficient resolution of proteins over a range of molecular weights. After electrophoresis, proteins were transferred to PVDF membranes using the VIX transfer system for optimal transfer efficiency. To block non-specific binding, PVDF membranes were immersed in 5% nonfat dry milk and gently vortexed at 80 rpm for 1 hour. After blocking, the membranes were washed with TBST and incubated with target-specific primary antibodies at 4°C for 8-12 hours under optimal conditions. After primary antibody incubation, the membranes were washed three times with TBST (5-15 minutes each) to remove unbound antibodies. They were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Protein signals were detected using an enhanced chemiluminescence (ECL) reagent. Membranes were processed in a chemiluminescence detection system at 4°C to ensure clear signal visualization and high band quality. Protein band intensity was quantified using ImageJ software, which allows densitometric analysis for accurate and reliable comparative protein expression studies. 3. Quantitative morphometric analysis of gastrocnemius muscle fibers Gastrocnemius muscle specimens were fixed in 4% paraformaldehyde for 24 hours to ensure optimal structural preservation. After fixation, specimens were subjected to graded ethanol dehydration followed by paraffin embedding for accurate sectioning. Thin 5-μm sections were prepared and stained with hematoxylin and eosin (H&E), a standard histological method that enhances the visualization of cellular and extracellular structures and provides a detailed depiction of muscle fiber architecture.
Institutions
- Xiamen UniversityFujian, Xiamen