Osteoporosis, a prevalent metabolic bone disease, increases fracture risk due to decreased bone mass and microarchitectural deterioration. While many treatments exist, they often involve patient exertion or undesirable side effects. Dietary intervention, particularly addressing high-fat diets (HFDs), is a promising approach. HFDs are associated with obesity and bone loss, potentially linked to gut microbiome imbalances. However, the role of obesity versus HFD itself in bone loss remains unclear. This study aimed to determine whether HFD-induced obesity or HFD alone caused bone loss and investigate the underlying mechanisms involving the gut microbiome, short-chain fatty acids (SCFAs), and regulatory T cells (Tregs). The gut microbiome plays a crucial role in regulating host physiology, including bone remodeling. SCFAs, produced by bacterial fermentation, influence bone metabolism and osteoclastogenesis. Tregs also play a role in bone health, but their involvement in HFD-induced bone loss is not fully understood. This study used HFD-induced obesity (HIO) and non-obesity (NO) mouse models to compare bone health under HFD and explore the role of the gut microbiome.

Existing literature shows a correlation between high-fat diets and bone loss, but the exact relationship is complex. Some studies suggest that high-fat diets, irrespective of obesity, negatively affect bone health, attributing this to increased fat intake. Others emphasize the role of obesity in bone loss. Previous research also highlights the gut microbiome's role in bone metabolism and its influence through immune modulation, endocrine signaling, and metabolites like SCFAs. Studies demonstrate that SCFAs enhance bone density and regulate osteoclastogenesis; and that Tregs have beneficial effects on bone loss in several animal models. However, the exact mechanisms of these factors in HFD-induced bone loss remained unclear before this study.

The study used male BALB/c mice, divided into chow diet (CD), HFD-induced obesity (HIO), and non-obese (NO) groups after 10 weeks of HFD feeding. NO mice had body weights within 5% of the CD group, while HIO mice were >20% heavier. Both HIO and NO groups were then fed HFD for another 10 weeks. Biomechanical testing (three-point bending) assessed tibia tenacity. Micro-CT analyzed bone microarchitecture (cortical and trabecular bone parameters). Immunohistochemistry quantified RANKL and OPG expression in tibiae. High-throughput sequencing analyzed gut microbiome composition in fecal samples. Fecal SCFA levels were measured using LC-MS. Metabolomic profiling of feces was also conducted using UPLC-MS. To investigate the role of SCFAs, one HIO group received SCFA supplementation. Fecal microbiota transplantation (FMT) from NO mice to HIO mice tested the microbiome's effect. Flow cytometry evaluated Treg populations in mesenteric lymph nodes. Ex vivo experiments using RAW264.7 macrophages co-cultured with T cells from different groups assessed the effects of Tregs on osteoclastogenesis. Real-time PCR quantified gene expression of relevant factors (Foxp3, IL-10, TGF-β1, HDACs, etc.).

HFD-induced obesity (HIO), but not HFD alone, caused bone loss. HIO mice showed reduced tibia tenacity, cortical bone density, trabecular bone volume, and increased trabecular separation compared to CD mice. NO mice maintained normal bone parameters despite long-term HFD. The gut microbiota differed significantly between HIO and NO mice, with NO mice exhibiting greater bacterial diversity and increased SCFA-producing bacteria. NO mice had higher fecal acetate and butyrate levels than HIO mice. SCFA treatment in HIO mice improved bone parameters. FMT from NO mice to HIO mice ameliorated bone loss and altered the gut microbiome in the recipients to resemble that of NO mice. NO mice showed significantly higher percentages of CD4+CD25+Foxp3+ Tregs and CD4+CD25+ T cells compared to HIO mice. T cells from NO mice inhibited osteoclast differentiation in RAW 264.7 macrophages ex vivo, reducing RANKL and TRAP expression and increasing IL-10 levels. FMT from NO mice increased Foxp3+ Tregs and IL-10 expression in recipient HIO mice. NO FMT reduced HDAC2 and HDAC7 expression and increased P300, Crebbp, and Pcaf expression in the colon of recipient mice. Obesity-related metabolites (purines) were significantly different between HIO and NO groups.

This study demonstrates that obesity, not simply HFD, is the crucial factor driving bone loss. The gut microbiome plays a pivotal role, with the NO microbiome's capacity to produce SCFAs being protective against bone loss. SCFAs, acting through FFAR2 and HDAC inhibition, promoted Treg cell proliferation, which inhibited osteoclastogenesis. This highlights the gut-bone axis and the potential of manipulating the microbiome to improve bone health. The differences observed in purine metabolites support the notion that the NO microbiome might improve bone health by affecting purine metabolism. These findings have significant implications for personalized nutrition strategies, suggesting that dietary interventions should account for individual microbiome variations.

This study clearly shows that HFD-induced obesity, rather than the HFD itself, is the primary driver of bone loss. The protective effect of the non-obese gut microbiome, mediated by SCFAs and Treg cells, offers a promising therapeutic avenue. Future research should focus on identifying specific bacterial species crucial for SCFA production and developing strategies to manipulate the gut microbiome for preventing or treating obesity-related bone loss.

The study used a mouse model, which might not fully translate to humans. More research is needed to confirm these findings in human populations. The study focused on bone loss in the tibia and femur; other skeletal sites may respond differently. While the mechanism is explored, further investigation is needed to completely understand the complex interactions between the gut microbiome, SCFAs, Tregs, and bone cells. The study did not examine specific genetic factors or other potential confounding variables influencing these effects.