Composition of gut microbiota involved in alleviation of dexamethasone-induced muscle atrophy by whey protein

Skeletal muscle atrophy, a reduction in muscle weight and function, is associated with increased morbidity and mortality. It's often caused by factors like glucocorticoid administration (e.g., dexamethasone, DEX), aging, and various diseases. DEX, a synthetic glucocorticoid, is widely used clinically but causes muscle atrophy by decreasing protein synthesis and increasing degradation. While dietary interventions, such as whey protein supplementation, can mitigate muscle atrophy, the mechanisms remain unclear beyond the provision of essential amino acids for muscle synthesis. The gut microbiota, a complex community of microorganisms in the gut, is increasingly recognized for its influence on various physiological processes, including muscle function. Previous research has linked gut microbiota composition to muscle function and whey protein's effectiveness in mitigating atrophy. This study aimed to investigate the role of gut microbiota in DEX-induced muscle atrophy and the potential of whey protein to ameliorate it by investigating the interplay between DEX treatment, gut microbial composition, and muscle function and weight, with a particular focus on identifying specific microbial signatures that can predict muscle health.

The literature extensively documents the detrimental effects of glucocorticoids, particularly DEX, on skeletal muscle. DEX reduces muscle protein synthesis while increasing protein degradation, leading to muscle wasting. While whey protein has shown promise in counteracting muscle atrophy by providing essential amino acids, this explanation is insufficient for conditions like DEX-induced atrophy, which aren't caused by nutrient deficiencies. Emerging evidence points towards the gut-muscle axis, highlighting the gut microbiota's influence on muscle function. Studies have shown that manipulating gut microbiota through antibiotics or fecal microbiota transplantation impacts muscle function and performance. Prior research from the authors' group demonstrated a link between gut microbiota composition and whey protein's efficacy in mitigating muscle atrophy in hematopoietic stem cell transplant patients. This study builds upon this foundation by investigating the direct impact of DEX on gut microbiota and the subsequent role of whey protein in restoring its balance and improving muscle health.

Six-week-old male C57BL/6J mice were divided into groups: a control group (DEX-), a DEX-treated group (DEX+), and DEX-treated groups with either natural recovery (D.D.) or whey protein intervention (D.W.). DEX was administered intraperitoneally for four weeks. After this period, the DEX+ mice were further divided into three groups for additional three weeks. The control group received saline injections. Body weight, food intake, and grip strength were monitored weekly. After 7 weeks of DEX treatment, muscle weight (gastrocnemius and soleus) was measured. Fecal samples were collected at week 8 for 16S rRNA gene sequencing to analyze gut microbiota composition. Alpha diversity (Chao1, Shannon, Observed_species, PD_whole_tree) was assessed. Principal coordinate analysis (PCOA) was used to visualize differences in gut microbiota structure. Spearman correlation analysis examined the relationship between gut microbiota composition and muscle function/weight. Stepwise regression models were built to identify enterotypes that could predict grip strength and gastrocnemius muscle weight, evaluating the models using R-squared and root mean square error. Statistical significance was set at p<0.05.

DEX administration significantly reduced body weight and grip strength in mice, indicating muscle dysfunction and atrophy. DEX also altered the gut microbiota, reducing alpha diversity, increasing the Firmicutes/Bacteroidota ratio, and leading to the loss of 1168 OTUs and gain of 480 new OTUs. Spearman correlation analysis revealed 28 bacterial genera associated with muscle strength and weight. Whey protein intervention in DEX-treated mice partially reversed the changes in gut microbiota induced by DEX and significantly improved muscle function and weight compared to the natural recovery group. Stepwise regression analyses identified two enterotypes that effectively predicted skeletal muscle function (grip strength) and weight (gastrocnemius muscle weight). These enterotypes contained key bacterial genera such as *Ileibacterium* and *Lachnospiraceae_UCG-001*, which showed a significant association with both muscle function and weight. Specifically, *Lachnospiraceae_UCG-001* and *Ileibacterium* were identified as key predictors in both models, suggesting their potential importance in regulating muscle health. Whey protein intervention significantly increased the relative abundance of *Lachnospiraceae_UCG-001* and *Ileibacterium*, whereas *Odoribacter* abundance was decreased.

This study demonstrates a strong link between DEX-induced muscle atrophy and alterations in gut microbiota composition. The observed changes in alpha diversity and the Firmicutes/Bacteroidota ratio suggest a disruption of gut microbial balance. Importantly, whey protein intervention effectively ameliorated muscle atrophy and modulated the gut microbiota toward a structure more similar to that of healthy mice. This suggests that whey protein's beneficial effects may partly be attributed to its ability to reshape the gut microbiome. The identified enterotypes, containing key bacterial species capable of producing SCFAs, further supports the role of gut microbiota in regulating muscle metabolism. The identified bacterial genera (*Ileibacterium* and *Lachnospiraceae_UCG-001*) warrant further investigation for their potential roles in muscle health and as therapeutic targets.

This study provides compelling evidence for the gut-muscle axis's role in DEX-induced muscle atrophy. Whey protein intervention effectively mitigates this atrophy, partly through its influence on gut microbiota composition. The identified predictive enterotypes offer potential for personalized interventions to combat muscle atrophy. Future research should focus on the mechanisms underlying the identified bacterial genera's influence on muscle metabolism, the specific SCFA produced by these genera and their downstream effects, and exploring the clinical translation of these findings for effective therapies targeting muscle atrophy.

The study utilized a mouse model, which may not perfectly reflect human physiology. The sample size was relatively small, limiting the generalizability of the findings. Further studies with larger sample sizes and human subjects are needed to validate these results. The specific mechanisms by which *Ileibacterium* and *Lachnospiraceae_UCG-001* influence muscle function need further elucidation.