Muscle integrity is important in obesity, but the development of compromised muscle integrity with obesity is poorly understood. Specifically, the relationships between compromised muscle integrity, systemic mediators, adipose tissue, and gut microbiota, have not been explored in detail or in an integrated manner following a short-term metabolic challenge. Based on the data from this study, we suggest that a link exists between HFS metabolic challenge and persistent increases in intramuscular fat, fibrosis, and CD68+ inflammatory cell number in the VL muscle by 3-days on an HFS diet, although the specific mediator(s) responsible remain to be determined. In contrast to the sustained alterations in the VL muscle, the dynamic alterations observed in local gene expression, systemic inflammation and the gut microbiota early in the obesity induction phase could represent a reciprocal, dynamic competition of perturbations, which ultimately fails. This failure may initiate the process leading to obesity and compromised muscle integrity, potentially setting the stage for which progressive, chronic musculoskeletal alterations with obesity may be established11. Although the present study was not designed to evaluate direct causal or mechanistic relationships between metabolic challenge and muscle integrity, the insight gained through this integrated investigation provides critical understanding for the role of obesity on altered muscle integrity.

Abnormal muscle repair is defined by sustained muscle fibrosis, which interferes with the appropriate healing of muscle tissue18. Notably, as previous studies have indicated that diet-induced obesity disrupts muscle repair processes19, the fibrosis observed in this study may be interpreted as HFS-induced muscle damage or disrupted repair. Here, the increased fibrosis observed by 3-days on the HFS diet is among the earliest reports of compromised muscle integrity with short-term metabolic challenge. Importantly, since fibrosis is a typical step in healthy muscle repair processes, the large variation in fibrosis in 7- and 14-day animals may reflect variation in responses among the animals to the severity of compromise in muscle with the HFS challenge. A tightening of such variation at 28-days suggests that a threshold may have been reached beyond which muscle repair is difficult. Therefore, 28-days on the HFS diet may prove to be an important time-point to determine whether muscle damage may still be reversible11.

Furthermore, little is known about how intramuscular fat may influence the muscle repair and regeneration process. However, ectopic fat accumulation in non-adipose tissues is a key feature of metabolic dysregulation3 and increased myocellular lipid content leads to increased lipid burden and insulin resistance3. In the present study, increased fibrosis at 3-days may result from increases in intramuscular fat deposition, which may signal the infiltration of pro-inflammatory cells, and they become activated. Additionally, VL fibrosis was associated with intramuscular constituents likely related to intramuscular fat (PPARγ) and resulting muscular inflammation (COX-2, iNOS), further implicating a link between intramuscular lipids and fibrosis. However, the present analysis of intramuscular fat did not differentiate between lipid location within the muscle (i.e. intramyofiber lipids, external muscle cell deposits, and non-muscle fat-containing cells) due to the small percentages of intramuscular fat detected in this short time frame, which is a limitation that will be addressed in future studies.

Moreover, dysregulated repair may be indicated by the increased presence of CD68+ cells that occurred by 3-days and was sustained over the 28-day metabolic challenge. MCP-1, a proinflammatory macrophage chemo attractant, was transiently increased in muscle after 7-days on the HFS diet, and despite these transient increases, increases in CD68+ cells compared to chow-fed control values were sustained. Although MCP-1 and macrophages both have known protective and regenerative effects in skeletal muscle20, overexpression of MCP-1 coupled with increases in macrophages in skeletal muscle can induce inflammation, lead to alterations in glucose metabolism5, are associated with the development of insulin resistance21, and may alter intramuscular fat cell function, inducing fat cell differentiation22. Although our animals did not display significant differences in fasting glucose or insulin levels at the early time points assessed, these data suggest that dynamic molecular changes in muscle inflammation can lead to sustained muscle damage, potentially before overt metabolic dysregulation is established.

Whether compromised muscle integrity is secondary to the dynamic fluctuations in the microbiota, and subsequent down-stream increases in systemic inflammation, remains to be confirmed. However, we did observe dynamic changes in the gut microbiota composition in HFS-fed animal as early as 3-days, which is consistent with short-term changes observed in both humans23 and rodents24. Here, it appears that certain microbial groups were more sensitive to the abrupt change in diet, as evidenced by significant deviations from the chow-fed control animals at earlier time-points. However, the gut microbial community is a complex ecosystem, where each microbial taxa may play a different role in establishing its overall composition25. Furthermore, studies indicate that while diet can induce rapid changes in gut microbiota composition, these alterations may be transient due to the high resiliency of the gut microbiota community composition, and can thus return to a stable state26. These notions are consistent not only with the observed patterns in each microbial group profiled here, but also with the PCA analysis, which indicates that the overall composition of the gut microbiota was most similar in animals exposed to HFS for longer time periods. Altogether, these data suggest that, upon initial exposure to the HFS diet, acute changes in gut microbiota occur, which then ultimately impact the overall composition observed at the 28-day time point.

There is considerable evidence in animal models that the gut microbiota affects host physiology, including muscle function16,27. Although the specific molecular mechanisms involved in crosstalk across the gut-microbiota-muscle axis16,27 and the relationship between muscle properties and the gut microbiota remain poorly understood28, IL-6, MCP-1 and TNF-α have been identified as potential mediators between the gut microbiota and downstream alterations in muscle function27,29. Circulating levels of these three pro-inflammatory cytokines, which have been reported to be associated with diet-induced changes in the gut microbiota, are linked with muscle atrophy, lower muscle mass and reduced muscle strength in humans and animals30, further linking gut microbiota, systemic inflammation, and compromised muscle integrity in the present study. Specifically, others have shown that high fat diet increases gut permeability, which allows bacterial lipopolysaccharide (LPS) translocation31. In turn, LPS binds to toll-like receptor-4, and induces circulating IL-6 and TNF-α32, which are reported to be associated with muscle atrophy, lower muscle mass, and reduced muscle strength in humans and animals30, and were increased at 3-days in the present study, further linking gut microbiota, systemic inflammation, and compromised muscle integrity in this study. Although we did not directly measure LPS in these animals, we suggest that inflammation resulting from increases in circulating IL-6, and TNF-α, potentially due to diet-induced dynamic changes in gut microbial composition, may recruit pro-inflammatory macrophages to muscle and adipose tissue, ultimately promoting infiltration of macrophages as early as 3-days in the muscle. Notably, the levels of CD68+ cells in VL muscle did not subside, even though both the changes in gut microbial composition, VL and adipose mRNA, and serum inflammatory markers fluctuated throughout the study. Taken together, these data suggest that even short-term exposure to a HFS diet may lead to lasting changes in the VL muscle. Finally, as the mechanistic link between the changes in gut microbiota and systemic alterations detected in the present study may involve increases in gut permeability and subsequent translocation of LPS across the intestinal barrier, these possibilities will be explored in future studies to facilitate a deeper understanding of the short-term changes described here.

In summary, we suggest that short-term metabolic challenge results in rapid compromise of VL muscle integrity and elevation in tissue-level inflammation, resulting from systemic inflammation and alterations in the gut microbiota following short-term exposure to a HFS diet. Obesity induction appears to be a dynamic process that includes compensation across tissues and systems. Intervention or modulation within this early period may be of interest to mitigate important sustained alterations in muscle that occur early in the obesity induction period.