Obesity, a global health concern, is linked to various metabolic complications affecting the entire body's metabolism. One significant complication is increased fat accumulation in bones, leading to heightened fracture risk. Obesity primarily stems from excessive caloric intake or an imbalanced diet high in saturated and trans-unsaturated fatty acids, resulting in increased body mass index (BMI) and complications impacting bone and fat metabolism. Studies have shown that obesity negatively affects bone quality and the phenotype of bone marrow stromal cells (BMSCs) due to increased adipogenesis and a senescent microenvironment, leading to increased bone fragility. Current treatments for obesity, type 2 diabetes, and bone fracture risk are challenging and often involve lifestyle changes like dietary interventions and increased physical activity. Omega-3 polyunsaturated fatty acids (omega-3 PUFAs), including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are essential dietary components with various health benefits. They act as natural hypolipidemics, reducing hepatic fat accumulation, ameliorating obesity-induced low-grade inflammation, increasing adiponectin plasma levels, and enhancing intestinal fatty acid oxidation. Previous research has indicated a positive effect of omega-3 PUFAs on bone health in different conditions, including osteoporosis, obesity, and aging. However, the impact of omega-3 PUFAs on BMSC metabolism and bone marrow adiposity (BMA) under obesogenic conditions remains inadequately studied. While some animal studies using high-fat diets supplemented with omega-3 PUFAs have shown positive effects on bone parameters, they often lacked focus on early intervention timepoints in younger animals, neglecting the early impact of metabolic disturbances on bone homeostasis and stem cell properties within the bone microenvironment. In vitro studies suggest that omega-3 PUFAs may induce changes in BMSC plasma membrane composition, promoting osteoblastic differentiation and inhibiting osteoclast differentiation. This study aimed to investigate whether omega-3 PUFA supplementation in a high-fat diet could mitigate the detrimental effects on bone microstructure, bone marrow adipose tissue (BMAT), and the molecular characteristics of BMSCs and osteoclasts in a young mouse model of diet-induced obesity.

Existing research highlights the detrimental effects of obesity on bone health, characterized by increased bone marrow adiposity and impaired bone microarchitecture. Studies have demonstrated a correlation between obesity and increased risk of fractures due to decreased bone density and strength. Several studies have explored the potential benefits of omega-3 polyunsaturated fatty acids (PUFAs) in mitigating these negative effects. Some research suggests that omega-3 PUFAs can improve bone mineral density, reduce inflammation, and positively influence bone cell differentiation. However, the precise mechanisms by which omega-3 PUFAs impact bone metabolism in the context of obesity remain unclear. Prior investigations have shown conflicting results regarding the effects of omega-3 PUFA supplementation on bone parameters in animal models, potentially due to variations in dietary composition, animal models, and duration of intervention. The current study aims to address these knowledge gaps by focusing on the early impact of omega-3 PUFA supplementation on bone and bone marrow cells in a well-established mouse model of diet-induced obesity.

Twelve-week-old male C57BL/6N mice were randomly assigned to three dietary groups for eight weeks: (1) normal diet (ND), (2) high-fat diet (HFD), and (3) HFD supplemented with omega-3 PUFAs (HFD+F). Body weight, food intake, glucose tolerance, and fasting insulin levels were monitored weekly. After eight weeks, mice were sacrificed, and various analyses were performed. Bone microarchitecture was assessed using micro-computed tomography (µCT) of the proximal tibia and L5 vertebrae. Bone strength was evaluated using a three-point bending test on femurs. Bone marrow adipose tissue (BMAT) volume was quantified using contrast-enhanced µCT (CECT) with Hexabrix as a contrast agent. Histomorphometric analysis of H&E-stained sections was also conducted to confirm BMAT volume. Global lipidomic and metabolomic analyses were performed on plasma, bone marrow, and bone powder samples using liquid chromatography-mass spectrometry (LC-MS). Bone marrow stromal cells (BMSCs) were isolated, and their proliferation, colony-forming units-fibroblast (CFU-f), adipogenic, and osteoblastic differentiation potential were assessed using various assays (Oil Red O staining, Alizarin Red staining, alkaline phosphatase activity, and gene expression analysis). Osteoclast (OC) differentiation capacity was evaluated using TRAP staining, TRACP activity, and gene expression analysis. Hematopoietic stem cells (HSCs) were isolated, and their insulin and lipopolysaccharide (LPS) responsiveness was assessed using western blot analysis. Finally, the bioenergetic profile of BMSCs was determined using the Seahorse XFe24 Analyzer. Statistical analyses included one-way ANOVA, unpaired t-tests, and post-hoc tests as appropriate.

Omega-3 PUFA supplementation significantly improved several key parameters: 1. **Metabolic Parameters:** The HFD group exhibited worsened metabolic parameters (body weight gain, glucose tolerance, and fasting insulinemia), while the HFD+F group showed improvements in these parameters compared to the HFD group. Food intake remained unaffected. 2. **Bone Microstructure and Strength:** µCT analysis revealed that omega-3 PUFA supplementation increased trabecular bone volume (Tb.BV/TV) and trabecular number (Tb.N) in the proximal tibia of the HFD+F group compared to the HFD group. In the L5 vertebrae, omega-3 PUFAs improved cortical porosity (Ct.Po), cortical thickness (Ct.Th), cortical bone volume (Ct.BV/TV), and cortical area fraction (B.Ar/T.Ar). Three-point bending tests demonstrated significantly stronger femora in HFD+F mice than in HFD or ND mice. 3. **Bone Marrow Adiposity:** CECT analysis showed a significant decrease in bone marrow adipocyte (BMAd) number and BMAT volume in the HFD+F group compared to the HFD group. This reduction was also confirmed by histomorphometric analysis of H&E-stained sections. 4. **Lipidomics and Metabolomics:** LC-MS analysis revealed significant differences in lipid and metabolite profiles across the three groups. The HFD+F group exhibited decreased levels of several lipid classes in plasma (triglycerides, diacylglycerols, cholesterol, ceramides, and phosphatidylcholines), while levels of DHA and EPA were increased in both plasma and bone marrow. 5. **BMSC Differentiation:** BMSCs from the HFD+F group showed increased CFU-f, indicating enhanced stem cell properties. Adipogenic differentiation was reduced, while osteoblastic differentiation was enhanced. Gene expression analysis confirmed these findings, showing decreased expression of adipogenic markers and increased expression of osteoblastic markers. Senescence markers (p53) were also downregulated in the HFD+F group. 6. **Osteoclast Differentiation:** While the HFD group showed increased osteoclast formation, omega-3 PUFA supplementation reduced osteoclast formation. In vitro studies further confirmed the inhibitory effect of omega-3 PUFAs on osteoclast differentiation. 7. **HSCs Insulin and Inflammatory Responsiveness:** HSCs from the HFD+F group exhibited decreased insulin responsiveness and reduced NF-κB activation upon LPS stimulation compared to the HFD group, suggesting reduced inflammation. 8. **BMSC Metabolism and Senescence:** BMSCs from the HFD+F group demonstrated a quiescent metabolic state (reduced glycolytic and respiratory activity) along with reduced senescence markers (β-galactosidase activity and ROS production). Gene expression analysis confirmed a decrease in senescence-associated secretory phenotype (SASP) markers.

This study provides compelling evidence for the beneficial effects of omega-3 PUFA supplementation on bone health in the context of obesity. The observed improvements in bone microstructure, mechanical properties, and reduced bone marrow adiposity strongly suggest that omega-3 PUFAs can mitigate the negative effects of high-fat diets on bone. The positive influence on BMSC differentiation, reduced osteoclast formation, and decreased senescence further support these findings. The improved metabolic parameters observed in the HFD+F group suggest a broader systemic benefit beyond the skeletal system. The observed changes in lipid and metabolite profiles further underscore the multifaceted effects of omega-3 PUFAs on metabolism. These results are consistent with previous studies highlighting the anti-inflammatory and anti-adipogenic properties of omega-3 PUFAs. The findings of this study offer a potential novel therapeutic strategy for managing metabolic bone diseases associated with obesity, emphasizing the importance of dietary interventions in maintaining bone health.

This study demonstrates that omega-3 PUFA supplementation effectively improves bone health in an obese mouse model by reducing bone marrow adiposity, enhancing osteoblast differentiation, inhibiting osteoclastogenesis, and decreasing cellular senescence. These findings highlight the potential of omega-3 PUFAs as a therapeutic strategy for metabolic bone diseases associated with obesity. Future research could explore the long-term effects of omega-3 PUFA supplementation, investigate the optimal dosage and duration of treatment, and evaluate the effects in different animal models and human populations.

The study utilized a relatively short intervention period (8 weeks) and only male mice. Future studies should investigate the long-term effects of omega-3 PUFA supplementation, explore the effects in female mice, and examine the results in larger cohorts to increase the generalizability of the findings. Furthermore, the study focused primarily on diet-induced obesity; future studies might explore other obesity models to ascertain the broad applicability of the findings. Although the study employed several advanced techniques for data collection and analysis, some limitations associated with animal model studies still exist.