Brown fat-specific mitoribosomal function is crucial for preventing cold exposure-induced bone loss

Brown fat-specific mitoribosomal function is crucial for preventing cold exposure-induced bone loss | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Brown fat-specific mitoribosomal function is crucial for preventing cold exposure-induced bone loss Jingwen Tian, Ji Sun Moon, Ha Thi Nga, Ho Yeop Lee, Thi Linh Nguyen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4539468/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jul, 2024 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 4 You are reading this latest preprint version Abstract This study investigates the relationship between ambient temperature, brown adipose tissue (BAT) function, and bone metabolism, revealing how cold exposure and BAT mitochondrial activity influence bone health. Utilizing ovariectomized (OVX) mice to model primary osteoporosis and BAT-specific mitochondrial dysfunction (BKO) mice, we explored the effects of housing temperature on bone density, immune modulation in bone marrow, and the protective role of BAT against bone loss. The results showed that cold exposure universally decreases bone mass, increases osteoclastogenesis, and shifts bone marrow T-cell populations, suggesting a key role of the immune system in bone remodeling under cold stress. Crucially, the thermogenic function of BAT, underpinned by mitochondrial oxidative phosphorylation, protected against bone loss. Impairments in BAT function, either through surgical removal or mitochondrial dysfunction, exacerbated bone loss in cold environments, highlighting the importance of BAT metabolic activity for bone health. Additionally, we found that cold-induced alterations in BAT function led to systemic metabolic changes, including increased long-chain fatty acid levels, which directly and indirectly affected osteoclast differentiation and activity. These findings point to a systemic mechanism via which environmental temperature and BAT metabolism are interconnected with bone physiology, offering new insights into the metabolic and environmental determinants of bone health. In summary, our study underscores the complex interactions between ambient temperature, BAT function, and bone health, suggesting that novel bone disease therapies could be developed by targeting metabolic and environmental factors. Further research in these pathways may provide innovative approaches for managing bone health amid changing environmental conditions and metabolic dysfunctions. Cold exposure bone loss brown adipose tissue mitochondria Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction External temperature, as an environmental factor, influences numerous physiological processes, necessitating continuous adaptation of organisms to variations in temperature. Temperature variations can impact the functioning of the nervous system[29], endocrine system[8], and musculoskeletal system.[1; 14] Exposure to warm conditions can influence developmental processes, as shown by increases in the growth of the femur[27] and promotion of a compact and structurally robust trabecular and cortical bone structures.[14] Heating one limb from an early age leads to the growth of extremities solely on the heated side.[28] Additionally, chondrocytes, when cultured in warm conditions, exhibit increased proliferation rates.[26] Recently, the link between ambient temperature and bone density has garnered significant scientific attention. However, the full impact of environmental temperature on bone balance remains unclear. Certain studies have suggested that low temperature may increase bone density.[1] Conversely, other research has indicated that low temperature may adversely affect bone volume (BV), exemplifying the contrasting impacts of low temperature on bone health.[14; 25] Exposure to cold temperatures is also known to influence the production and release of neurotransmitters involved in the regulation of skeletal metabolism and endocrine functions.[19; 33] Neuropeptide Y (NPY) has been reported to increase energy expenditure, upregulate uncoupling protein 1 (UCP-1) expression, and contribute to bone loss during cold exposure.[33] Activation of sympathetic nerves by low temperatures triggers β-adrenergic receptors, leading to the initiation of bone resorption processes by osteoclasts.[4; 10] Moreover, these activated sympathetic nerves indirectly impact bone mass by modulating the expression of bone morphogenetic protein 8b (BMP8b) and parathyroid hormone (PTH).[10; 11] Additionally, a recent paper reported that thermoneutral housing of mice mitigates bone loss induced by ovariectomy through the regulation of the gut microbiome environment.[6] Osteoporosis, a prevalent metabolic bone condition, is characterized by reduced bone density and the weakening of bone microarchitecture,[31] resulting in more fragile bones and an increased likelihood of fractures. The most common type of primary osteoporosis occurs as a result of post-menopausal estrogen deficiency,[23] and as such, it is exceedingly common in older women but can also occur in men. The impact of living temperature conditions during post-development and late adulthood, both in healthy individuals and those with osteoporotic conditions, on bone health, remodeling, and physiological processes remains uncertain. Maintaining a constant body temperature in a changing temperature environment is critical for survival. When exposed to low temperatures, mammals increase oxidative metabolism for heat production. BAT is the main site of non-shivering thermogenesis in mammals. BAT activation is a complex physiological response to cold exposure, in which norepinephrine-mediated signaling promotes lipolysis, releasing free fatty acids and glycerol. These substrates fuel mitochondrial beta-oxidation and, via UCP-1, facilitate thermogenesis instead of ATP production,[30] thereby increasing energy expenditure and contributing to metabolic health. Brown adipocytes are characterized by their abundant mitochondria, which possess a high capacity for oxidation, and contain UCP-1 within their inner membranes.[24] BAT mitochondrial oxidative activity is increased by cold exposure.[18] UCP-1, which is unique to brown adipocytes, disrupts the link between the respiratory chain and oxidative phosphorylation, leading to increased oxidation rates and the conversion of metabolic energy into heat.[3] BAT volume has been reported to correlate with bone mass,[21] to influence positively bone mass, and to serve as a reliable indicator of the structural integrity of the femur.[2] A previous study reported that BAT-deficient mice show a marked reduction in bone formation.[22] Additionally, studies in human have identified brown adipose tissue as a significant independent factor influencing bone mass,[1; 20] and in mice, BAT has been shown to rescue bone loss induced by cold exposure at an early time point.[9] In the current study, we explored BAT function-related bone phenotypes using BAT-specific mitochondrial oxidative phosphorylation (OxPhos) deficient mice. Materials and Methods Mice Ucp1-cre transgenic (Tg [ Ucp-Cre ]1Evdr) mice were purchased from Jackson Laboratory, and were backcrossed to C57BL/6J background mice. Floxed Crif1 ( Crif1 f/f ) mice were generated as previously described.[16] The mice in this study were kept in a specific pathogen-free environment at the Preclinical Research Center of Chungnam National University Hospital and were housed under standard conditions, which included a cycle of 12 h of light followed by 12 h of darkness, a stable room temperature of 22°C, and a relative humidity of between 40–60%. Their diet consisted of Teklad global chow with 18% protein content (2918C, ENVIGO). Severe cold and thermoneutral housing conditions were performed at 14°C and 30°C, respectively, in a light and humidity-controlled chamber. All experimental procedures complied with the guidelines of Institutional Animal Care and Use Committees (IACUCs) Micro-CT analysis. Micro-CT was performed on vertebrae and long bones using SkyScan 1173 (SkyScan, Belgium) with 8 µm resolution. All bone morphometric parameters were calculated three-dimensionally with CTan and CTvox version 1.6, which was used to measure BV, total volume, BV/TV, bone surface, bone surface density, trabecular thickness, and trabecular separation. All bone micro-CT nomenclature followed the guidelines of the American Society for Bone and Mineral Research (ASBMR). Statistical analysis All Statistical analyses were conducted using GraphPad Prism software (version 9, Dotmatics, San Diego, USA). Data were expressed as the mean ± SD. Unpaired Student's t-tests and one-way ANOVA followed by Scheffe's post-hoc test were used to determine statistical significance, with a p-value of less than 0.05 considered significant. Results Low temperature accelerates bone loss in mice Temperatures of 28–33°C are regarded as thermoneutral housing conditions for mice based on studies of mouse metabolic rates at different temperatures. Typical room temperatures for housing mice can be considered mild cold conditions, whereas temperatures below 16°C are viewed as severe cold conditions, necessitating shivering thermogenesis.[12] To explore the effects of housing temperature on bone mass homeostasis, we employed micro-computed tomography (micro-CT) analysis to evaluate the bone structural features of SHAM and ovariectomized (OVX) mice, a commonly used primary osteoporosis model. After performing OVX and SHAM surgery on 8-week-old mice, the mice were maintained at 22°C, corresponding to standard room temperature housing conditions, and 30°C, corresponding to thermoneutral housing conditions, for 12 weeks, and the experiments were performed when the mice were 20 weeks old. Micro-CT analysis was conducted to assess cortical and trabecular bone architecture of SHAM and OVX mice housed under room temperature conditions and those housed under thermoneutral conditions. As expected, in trabecular bone, thermoneutral housing conditions significantly increased bone mineral density (BMD), BV, and trabecular number in OVX mice but not in SHAM mice (Fig. 1 A and 1 B). In cortical bone, housing temperature had no effect on these parameters in either SHAM or OVX mice (Supplementary Fig. 1A and 1B). Next, we examined the bone phenotypes under severe cold housing conditions (14°C). The SHAM mice housed under these conditions showed decreases in trabecular and cortical bone parameters compared with those housed under room temperature conditions. In OVX mice housed under severe cold conditions, trabecular BMD was significantly lower than in those housed under room temperature conditions (Supplementary Fig. 2A and 2B). Tartrate-resistant acid phosphatase (TRAP) staining revealed that the number of mature osteoclasts was significantly higher in OVX mice than in SHAM mice and significantly higher in OVX mice housed at 22°C than in those housed at 30°C (Fig. 1 C and 1 D). However, there was no significant difference in the serum level of P1NP, a bone formation marker, between mice housed at 22°C and those housed at 30°C (Fig. 1 E). Collectively, exposure to lower temperature appears to induce bone loss accompanied by an increase in the number of osteoclasts. Temperature-dependent modulation of bone marrow T-cells and BAT function Next, we investigated how housing temperature affects bone marrow immune cell phenotypes, which is an important factor in the initiation of bone resorption [32]. To address this issue, we examined the expression of osteoclastogenic cytokines in bone marrow cells from mice housed at 22°C and 30°C. We observed a decrease in the populations of CD4 + CD25 + Foxp3 + regulatory T-cells (Tregs) in both OVX and SHAM mice at 22°C. Previous studies showed that Tregs produce RANKL, which is important for osteoclastogenesis and is linked to the bone damage observed in inflammatory arthritis [17]. We found that the expression of RANKL (CD254 + ) in Tregs was higher in OVX mice housed at 22°C than in those housed at 30°C. The expression of IFN-γ, a cytokine known to inhibit osteoclast differentiation, was lower in the CD4 + cells of OVX mice housed at 22°C than in those housed at 30°C. The expression of IL-17A + was higher in CD4 + cells of OVX mice than in those of SHAM mice, irrespective of housing temperature (Fig. 2 A and 2 B). These changes subsequently regulate the proliferation of osteoclasts. Additionally, we examined changes in BAT, an organ known for its temperature sensitivity. As reported previously, at lower temperatures, BAT exhibited significantly higher expression of UCP-1 and mitochondrial OxPhos complex subunits, including complex I (NDUFB8), II (SDHB), and IV (COX4) (Fig. 2 C and 2 D). Immunohistochemistry also revealed higher UCP-1 and succinate dehydrogenase (SDH) expression in BAT at lower temperatures in SHAM mice (Fig. 2 E). Impact of BAT removal on bone loss Next, we investigated whether BAT is directly involved in bone loss caused by cold exposure. At 8 weeks of age, mice were randomly assigned to undergo either a sham operation (SHAM mice) or surgical removal of interscapular BAT (BAT-deficient mice). Subsequently, the SHAM and BAT mice were housed at temperatures of either 22°C or 14°C for 12 weeks. The BAT-deficient mice housed at either of these temperatures did not show BAT regeneration at the end of the 12-week period. (Fig. 3 A). The BAT mice housed at 22°C showed decreases in trabecular BMD, trabecular BV, and trabecular percent BV, and an increase in trabecular separation. Cortical BV was also decreased in the cortical bone region. The BAT mice housed at 14°C also tended to show decreases in bone parameters, but the decreases were not statistically significant (Fig. 3 B and 3 C), with the exception of trabecular BMD. These findings lend support to the hypothesis that surgical removal of BAT exacerbates bone loss induced by cold exposure. BAT-specific mitochondrial OxPhos dysfunctional mouse model Mice with a tissue-specific deficiency of Crif1 (CR6-interacing factor 1), which encodes a protein of the large subunit of the mitochondrial ribosome, have markedly impaired mitoribosome-mediated translation [7; 15; 16]. In this study, to determine the impact of BAT mitochondrial OxPhos dysfunction on the bone, we generated BAT-specific Crif1 knockout (BKO) mice through selective disruption of Crif1 in brown adipocytes using the Cre-loxP system. Crif1 -floxed ( Crif1 f/f ) mice were bred with Ucp1-Cre transgenic mice, resulting in the deletion of exon 2 of the Crif1 gene (Fig. 4 A). Crif1 deficiency resulted in reduced translation of CRIF1, OxPhos subunits, including complex I (NDUFB8), complex II (SDHB), complex III (UQCRC2), and complex IV (MTCO1), and decreased UCP-1 expression in mice housed under chronic cold (22°C) conditions (Fig. 4 B and 4 C). Blue native-PAGE (BN-PAGE) analysis of mitochondria isolated from BAT revealed reduced levels of native OxPhos complexes I and III (Fig. 4 D). Immunohistochemistry staining showed that BAT from the Crif1 -knockout had reduced succinate dehydrogenase (SDH) expression, confirming reduced mitochondrial oxidative phosphorylation in the OxPhos dysfunctional mice, and reduced UCP-1 expression (Fig. 4 E and 4 F). Taken together, these findings imply that Crif1 deficiency in BAT sufficiently impairs OxPhos dysfunction in mice. Mitochondrial oxidative phosphorylation dysfunction in BAT accelerates bone loss caused by chronic cold stress Loss of mitochondrial function in BAT is expected to result in multiple phenotypes upon cold exposure. Thus, we hypothesized that cold-induced activation of BAT would be compromised in BAT with mitochondrial oxidative phosphorylation dysfunction, leading to bone loss. To validate this hypothesis, we assessed the bone parameters of BKO mice at 20 weeks of age using micro-CT. In the trabecular region, the BKO mice housed at 22°C exhibited significant decreases in BMD, BV, percent BV, trabecular number and thickness, and an increase in trabecular separation (Fig. 5 A and 5 B) compared with control mice housed under the same condition. Additionally, cortical BMD, BV, and thickness (Fig. 5 A and 5 B) were lower in these mice than in the control mice. Furthermore, BKO mice maintained at 30°C for 12 weeks started to show reversal of bone loss in the trabecular region at 8 weeks of age (Fig. 5 A and 5 B). Additionally, while there was no pronounced increase in BMD in cortical bone of BKO mice maintained at 30°C for 12 weeks, there was a discernible improvement in cortical BV and thickness (Fig. 5 A and 5 B). At a housing temperature of 22°C, BKO mice consistently displayed lower trabecular BMD and BV (Supplementary Fig. 3A and 3B) than control mice. Moreover, von Kossa staining showed decreased bone parameters not only in the femur but also in the vertebrae (Supplementary Fig. 3C). Taken together, these results indicate that BKO mice exhibit bone loss when exposed to chronic cold stress (22°C), but under thermoneutral conditions (30°C) have bone parameters similar to those of wild-type mice housed under the same conditions. This suggests that the activation of BAT mitochondria during cold exposure plays a pivotal role in maintaining bone mass. Next, we investigated whether mitochondrial dysfunction in BAT affects the bone marrow immune environment, which is a critical determinant for the initiation of bone resorption, and further determined whether mitochondrial OxPhos dysfunction in BAT triggers osteoclastogenesis in bone marrow. To do this, we quantified various T-cell populations in the BM of control and BKO mice by flow cytometry analysis. We found that at 20 weeks of age, Treg populations were significantly larger in the bone marrow of BKO mice (Fig. 5 C) than in that of control mice, Additionally, a subset of the Treg cell population expressing RANKL was also higher in BKO mice than in control mice (Fig. 5 C). The serum level of RANKL was also increased in these mice (Fig. 5 D). Furthermore, in the CD4 + T-cell population, IFN-γ expression, which is known to inhibit osteoclast differentiation, was lower in BKO mice than in control mice (Fig. 5 C). Thus, at low temperatures (22°C) requiring thermogenesis, BKO mice with BAT mitochondrial dysfunction have a bone marrow immune environment that favors osteoclastogenesis. Under thermoneutral conditions (30°C), the expression of RANKL in Treg cells and IFN-γ in CD4 + cells was lower than that in the controls (Fig. 5 C). Alterations in lipid composition within thermogenic adipose tissue and corresponding metabolite variations in serum Brown fat tissue is a site of active lipid catabolism, resulting in the release fatty acids for energy utilization via lipolysis. These fatty acids fuel thermogenesis, a process in which energy is not converted into ATP but instead released as heat. This is critical for body temperature regulation, especially during cold stress. This thermogenic response is orchestrated by UCP-1, which is prevalent in the mitochondrial membrane of brown fat cells and disrupts the typical pathway of oxidative phosphorylation.[5] In this study, we observed a decrease in the lipid area within BAT when the mice were housed at low temperatures (22°C and 14°C) (Supplementary Fig. 4A and 4B), Furthermore, Western blot analysis confirmed that cold temperature housing conditions activate lipolysis (Supplementary Fig. 4C and 4D), suggesting that BAT may counteract low temperatures by inducing thermogenesis using the energy derived from lipolysis. Next, we hypothesized that BAT in BKO mice would exhibit impaired lipolysis and fatty acid oxidation. To test this hypothesis, we examined the BAT phenotype of BKO mice. The BKO mice had larger lipid areas in BAT and larger lipid droplet sizes in inguinal white adipose tissue (iWAT) (Fig. 6 A and 6 B). Additionally, Western blot analysis revealed reduced lipolysis in BAT (Fig. 6 C and 6 D) and RT-PCR revealed reduced expression of genes controlling fatty acid oxidation ( Cpt1a, Cpt1b, Acadm, Ppara , and Pgc1a ) (Fig. 6 E). Furthermore, BKO mice had elevated serum levels of free fatty acids (Fig. 6 F), which may be attributed to decreased lipolysis and fatty acid oxidation in these mice. As shown in Fig. 6 G, the increased metabolites in the BAT and serum of BKO mice were predominantly fatty acids, with a notable increase in long-chain fatty acid levels in serum. Collectively, dysfunctional BAT mitochondria impair BAT fatty acid oxidation, leading to elevated long-chain fatty acids in the bloodstream. Long-chain fatty acids activate osteoclasts and promote T-cell osteoclastogenic transformation Reductions in lipolysis and fatty acid oxidation in BAT increase serum free fatty acids levels and decrease bone mass in BKO mice, suggesting that serum free fatty acids may directly or indirectly influence bone marrow cells. To determine the effects of fatty acids on bone marrow cells, we treated various types of bone marrow cells with different fatty acids. First, we treated bone marrow-derived osteoclasts with short-chain fatty acids (propionic acid) and long-chain fatty acids (palmitic acid). TRAP staining revealed no changes in the number of TRAP-positive osteoclasts after treatment with propionic acid compared with treatment with vehicle; however, a marked increase in the number of TRAP-positive osteoclasts was observed when the osteoclasts were treated with palmitic acid (Fig. 7 A and 7 B). Next, we examined for potential changes in bone marrow immune cell phenotypes following treatment with long-chain fatty acids by performing flow cytometry of bone marrow immune cells treated with palmitic acid for 24 h. Upon treatment with palmitic acid, the number of RANKL-expressing Tregs increased dose-dependently with the concentration of palmitic acid (50 µM and 100 µM), whereas the number of IFN-γ expressing CD4 + T cells significantly decreased compared with the vehicle-treated control cells (Fig. 7 B and 7 C). Taken together, these results suggest that, among the metabolites altered by the reduced lipolysis or fatty acid oxidation in BAT, long-chain fatty acids (palmitic acid) directly or indirectly regulate osteoclast differentiation, potentially leading to bone loss. Discussion The relationship between ambient temperature and bone physiology is a complex and multifaceted one, with a growing body of evidence suggesting that environmental factors play a significant role in the regulation of bone metabolism. This study reveals that temperature is a critical determinant of bone remodeling, which has profound implications for our understanding of bone health and disease management. The impact of temperature on bone physiology can also be seen through the lens of thermoregulatory metabolism. Mild cold stress, common in standard mouse housing conditions, is known to affect tumor growth rates, CD8 + T-cell and dendritic cell function, and the activity of immunosuppressive cells.[13] This underscores the importance of considering environmental factors when studying bone physiology and its interactions with the immune system. Our study also explored the temperature-dependent changes in bone marrow T-cell populations, shedding light on the immune-mediated mechanisms that may contribute to bone remodeling. We observed an increase in Tregs, which are known to suppress osteoclastogenesis, at thermoneutral temperatures (Fig. 2 ). This suggests that a warmer ambient temperature may protect against bone loss by modulating the immune environment within bone marrow. Conversely, at low temperatures, we noted an increase in the pro-resorptive T-cell populations (Fig. 2 ), which could contribute to the increased bone resorption observed at these temperatures. The results potentially highlight the importance of immune system interactions in mediating the effect of temperature on bone density. An intriguing aspect of our study is the relationship between BAT activity and bone mass. BAT is known to play an important role in thermogenesis, the process via which heat is produced in organisms. Brown adipocytes are rich in mitochondria, which have a high oxidative capacity and contain UCP-1 in their inner membrane,[24] which is particularly active during cold exposure [18]. Numerous studies have reported a positive relationship between BAT activity and bone mass [1; 2; 20–22]. However, research into the mechanisms underlying the relationship between BAT and bone mass remains limited. Our data suggest that BAT activity correlates positively with bone mass, indicating that BAT protects against bone loss. This is further corroborated by the observation that mice lacking BAT have a reduced bone mass (Fig. 3 ). The thermogenic function of BAT, primarily driven by UCP-1 and mitochondrial oxidative phosphorylation, may be a key part of this protective mechanism. This is not only due to the thermogenerative properties of BAT but also to the metabolic activity associated with thermogenesis, which appears to have a systemic effect on bone tissue. Furthermore, our research suggests that BAT mitochondrial functions are crucial for bone health, particularly under cold stress conditions. Mice with compromised BAT mitochondrial functions exhibited accelerated bone loss when exposed to the cold, but not when exposed to thermoneutral conditions (Fig. 5 ). This underscores the importance of BAT mitochondrial integrity for the preservation of bone mass during environmental stress. The metabolic mechanisms that mediate the effects of temperature variations on bone physiology are multifaceted. Our research suggests that lipolysis and β-oxidation in BAT play a pivotal role in modulating the levels of systemic metabolites. Lipolysis, a critical process for cold-induced thermogenesis, is intricately connected to bone metabolism through alterations in metabolite profiles. We found that reduced mitochondrial function in BKO mice was linked to decreases in lipolysis and fatty acid oxidation, leading to an increase in fatty acid-based metabolites (Fig. 6 ). Furthermore, among the increased metabolites, long-chain fatty acids not only increased the differentiation of osteoclasts as evidenced by increased TRAP staining (Fig. 7 A and 7 B), but also induced changes in cytokine expression in T cells, such as RANKL expression in Tregs and IFN-γ expression (Fig. 7 ), thereby potentially exerting indirect control over osteoclast regulation. However, our study is not without its limitations. Although we observed that housing temperature affected bone mass and BAT confers protection against bone loss induced by cold exposure, the mechanisms responsible for cold-induced bone loss were not clearly identified in this study. In conclusion, chronic cold exposure is associated with alterations in immune cells and is linked to bone loss. BAT plays a crucial role in protecting against bone loss induced by cold exposure, and this protective effect of BAT is linked to mitochondrial fatty acid oxidation and lipolysis-driven thermogenesis, as well as the consumption of long-chain fatty acids. Mitochondrial dysfunction in BAT is known to lead to an increase in systemic long-chain fatty acid levels, Our in vitro experiments support these findings, indicating that long-chain fatty acids can directly and indirectly upregulate osteoclasts. in turn promoting osteoclastogenic T-cell activity and bone loss. Declarations Author Contributions JT and HSY designed the study and wrote the manuscript. JT, JSM, HYL, and DS performed in vitro and in vivo experiments. HTN, TLN, and HJJ helped with data collection and assembly. MS and JHL provided helpful suggestions for project design and comments for the improvement of the manuscript. Funding This work was supported by a grant from the Korea Health Technology R&D Project, through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HR22C1734 and HI23C153400). HSY was supported by the Basic Science Research Program, through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning, Korea (NRF-2023R1A2C3006220 and NRF-2021R1A5A8029876). Data Availability All data generated or analyzed during this study are included in this published article and its supplementary information files. All primary data will be shared by the lead contact upon request. Acknowledgements Not applicable Conflict of Interest The authors declare no competing interests. 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Int J Obes Suppl 5(Suppl 1):S23-27 https://doi.org/10.1038/ijosup.2015.7 Motyl KJ, Bishop KA, DeMambro VE, Bornstein SA, Le P, Kawai M, Lotinun S, Horowitz MC, Baron R, Bouxsein ML, Rosen CJ (2013) Altered thermogenesis and impaired bone remodeling in Misty mice. J Bone Miner Res 28(9):1885-1897 https://doi.org/10.1002/jbmr.1943 Reginster JY, Burlet N (2006) Osteoporosis: a still increasing prevalence. Bone 38(2 Suppl 1):S4-9 https://doi.org/10.1016/j.bone.2005.11.024 Ricquier D, Bouillaud F (2000) Mitochondrial uncoupling proteins: from mitochondria to the regulation of energy balance. J Physiol 529 Pt 1(Pt 1):3-10 https://doi.org/10.1111/j.1469-7793.2000.00003.x Robbins A, Tom C, Cosman MN, Moursi C, Shipp L, Spencer TM, Brash T, Devlin MJ (2018) Low temperature decreases bone mass in mice: Implications for humans. Am J Phys Anthropol 167(3):557-568 https://doi.org/10.1002/ajpa.23684 Serrat MA (2014) Environmental temperature impact on bone and cartilage growth. Compr Physiol 4(2):621-655 https://doi.org/10.1002/cphy.c130023 Serrat MA, King D, Lovejoy CO (2008) Temperature regulates limb length in homeotherms by directly modulating cartilage growth. Proc Natl Acad Sci U S A 105(49):19348-19353 https://doi.org/10.1073/pnas.0803319105 Serrat MA, Schlierf TJ, Efaw ML, Shuler FD, Godby J, Stanko LM, Tamski HL (2015) Unilateral heat accelerates bone elongation and lengthens extremities of growing mice. J Orthop Res 33(5):692-698 https://doi.org/10.1002/jor.22812 Shevchuk NA (2008) Adapted cold shower as a potential treatment for depression. Med Hypotheses 70(5):995-1001 https://doi.org/10.1016/j.mehy.2007.04.052 Sidossis L, Kajimura S (2015) Brown and beige fat in humans: thermogenic adipocytes that control energy and glucose homeostasis. J Clin Invest 125(2):478-486 https://doi.org/10.1172/jci78362 Sözen T, Özışık L, Başaran N (2017) An overview and management of osteoporosis. Eur J Rheumatol 4(1):46-56 https://doi.org/10.5152/eurjrheum.2016.048 Tian J, Chung HK, Moon JS, Nga HT, Lee HY, Kim JT, Chang JY, Kang SG, Ryu D, Che X, Choi JY, Tsukasaki M, Sasako T, Lee SH, Shong M, Yi HS (2022) Skeletal muscle mitoribosomal defects are linked to low bone mass caused by bone marrow inflammation in male mice. J Cachexia Sarcopenia Muscle 13(3):1785-1799 https://doi.org/10.1002/jcsm.12975 Wee NKY, Nguyen AD, Enriquez RF, Zhang L, Herzog H, Baldock PA (2020) Neuropeptide Y Regulation of Energy Partitioning and Bone Mass During Cold Exposure. Calcif Tissue Int 107(5):510-523 https://doi.org/10.1007/s00223-020-00745-9 Supplementary Files Supplementaryinfomation.docx Cite Share Download PDF Status: Published Journal Publication published 27 Jul, 2024 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Major Revision 22 May, 2024 Reviewers agreed at journal 28 Apr, 2024 Reviewers invited by journal 28 Apr, 2024 First submitted to journal 17 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4539468","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":311298777,"identity":"4487dc19-b23e-4720-80f3-fa1bc7b4e890","order_by":0,"name":"Jingwen Tian","email":"","orcid":"","institution":"Chungnam National University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingwen","middleName":"","lastName":"Tian","suffix":""},{"id":311298778,"identity":"43ce6488-bdfc-4cb7-96a0-67aef0f594d6","order_by":1,"name":"Ji Sun Moon","email":"","orcid":"","institution":"Chungnam National University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ji","middleName":"Sun","lastName":"Moon","suffix":""},{"id":311298779,"identity":"6159f7fb-93d4-4aab-b238-abbbc9fb928f","order_by":2,"name":"Ha Thi Nga","email":"","orcid":"","institution":"Chungnam National University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ha","middleName":"Thi","lastName":"Nga","suffix":""},{"id":311298780,"identity":"b6cda4a1-1c43-45c2-a895-fd87228f966e","order_by":3,"name":"Ho Yeop Lee","email":"","orcid":"","institution":"Chungnam National University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ho","middleName":"Yeop","lastName":"Lee","suffix":""},{"id":311298781,"identity":"7bef80c3-8a50-415e-9ada-d4b76d378f42","order_by":4,"name":"Thi Linh Nguyen","email":"","orcid":"","institution":"Chungnam National University School of 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Yi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBACxgYGNiCVwMAPE5EgWotkA7FagACixeAAsVqYp5099uBDRZq98fGzxyQYauwYJGcfwK+FcXZeuuGMMzmJ287kpUkwHEtmkOZLIKQlx0yat60iwewGj5kEA9sBBjkeAg6DabE3ngHS8o94LTmMGySAWhjbDjBIE9YC9ktaItA/xhaJfck8kj0EtBjOzgWFWLI9f/sZwxsfvtnJSZwhpKUB2R0JDAyEnMXAIE+EmlEwCkbBKBjpAABmVzmC/y+B/QAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-3767-1954","institution":"Chungnam National University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hyon-Seung","middleName":"","lastName":"Yi","suffix":""}],"badges":[],"createdAt":"2024-06-06 10:35:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4539468/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4539468/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-024-05347-4","type":"published","date":"2024-07-27T16:15:59+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59269727,"identity":"f0955049-4374-40a2-b3c7-fe38db44a911","added_by":"auto","created_at":"2024-06-28 12:19:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":316087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAggravation of ovariectomy-induced bone loss by low temperature housing conditions.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Illustrative micro-CT images showing the trabecular areas in the lower section of the femur. \u003cstrong\u003eB\u003c/strong\u003e Micro-CT measurement of trabecular bone mineral density (Tb.BMD); trabecular bone volume (Tb.BV); trabecular bone volume/tissue volume (Tb.BV/TV); trabecular bone surface/tissue volume (Tb.BS/TV); trabecular number Tb.N; and trabecular separation Tb.Sp \u003cstrong\u003eC,D\u003c/strong\u003e TRAP staining for osteoclasts and statistical analysis of osteoclast number. E, Serum levels of P1NP. The results were expressed as the mean ± SD. Statistical significance was estimated using unpaired t-tests. *, P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4539468/v1/cb4374d6aea19e070819a24c.png"},{"id":59270167,"identity":"99d740b6-4f84-4178-bb5a-49fe9665f981","added_by":"auto","created_at":"2024-06-28 12:27:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":376689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemperature-mediated changes in osteoclastogenic bone marrow T cells and brown adipose tissue (BAT).\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Representative contour plots from flow cytometry for regulatory T-cells (Treg; CD4+ CD25+ FOXP3+); RANKL (CD254) producing Treg cells; and IFN-γ or IL-17A-producing CD4+ cells from bone marrow. \u003cstrong\u003eB\u003c/strong\u003e Statistical analysis of phenotypes defined by flow cytometry. \u003cstrong\u003eC,D\u003c/strong\u003eRepresentative western blots and band density measurements of UCP-1 and OxPhos complex subunits in the BAT isolated from mice housed for 12 weeks at 22°C or 30°C. \u003cstrong\u003eE\u003c/strong\u003e Representative immunohistochemical images of UCP-1 and SDH staining of BAT from mice housed at different temperatures. Scale bar, 50 μm. Data were expressed as the mean ± SD. Statistical significance was estimated using unpaired t-tests. *, P \u0026lt; 0.05, **, P \u0026lt; 0.01 ***, P\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4539468/v1/57396b2f5c07a20a439f8c0a.png"},{"id":59269729,"identity":"67e9ee1d-17ee-48c3-9520-7d79c93a0c06","added_by":"auto","created_at":"2024-06-28 12:19:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":314333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRemoval of interscapular BAT promotes bone loss.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Images of mice at 12 weeks after post-surgical removal of BAT. \u003cstrong\u003eB\u003c/strong\u003e Representative images of micro-CT of cortical and trabecular regions in the distal femur. \u003cstrong\u003eC\u003c/strong\u003e Measurement of Tb.BMD, Tb.BV, Tb.BV/TV, Tb.N, Tb.Sp, Ct.BMD, Ct.BV, and Ct.Th in the femurofSHAM and BAT-deficient mice housed at 22°C or 14°C for 12 weeks. Statistical significance was estimated using unpaired t-tests. *, P \u0026lt; 0.05, **, P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4539468/v1/4446ac2e9e6751038bc6be75.png"},{"id":59270168,"identity":"1e78a043-20ad-49b9-9152-6eae4ce76534","added_by":"auto","created_at":"2024-06-28 12:27:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":296316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBAT-specific mitochondrial dysfunction (BKO) mice\u003c/strong\u003e \u003cstrong\u003eshow impairment of OxPhos.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e BKO mice generated by knockout of \u003cem\u003eCrif1\u003c/em\u003e in brown adipose tissue using UCP-1-cre mice and the \u003cem\u003eCre-loxP\u003c/em\u003esystem. \u003cstrong\u003eB,C\u003c/strong\u003e Immunoblotting of OxPhos complex subunits and UCP-1 in BAT isolated from control and BKO mice housed at 22°C at 20 weeks old. \u003cstrong\u003eD\u003c/strong\u003e Representative BN-PAGE image of the assembled OxPhos complex in BAT tissue. \u003cstrong\u003eE,F \u003c/strong\u003eRepresentative immunohistochemical images of UCP-1 and SDH staining in BAT paraffin sections. Scale bar, 50 μm. The statistical relevance of the findings was determined using unpaired t-tests. *, P \u0026lt; 0.05, **, P \u0026lt; 0.01 ***, P\u0026lt; 0.001. The analysis was conducted in comparison with the specified reference group.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4539468/v1/aed8c7a3c2b709f7a3856694.png"},{"id":59269735,"identity":"2a69a95c-1abb-4f30-a09a-c5d43561543b","added_by":"auto","created_at":"2024-06-28 12:19:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":492255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBAT-specific mitochondrial dysfunction (BKO) mice exposed to cold stress exhibit bone loss and an osteoclastogenic bone marrow T-cell phenotype.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Micro-CT scans showing the cortical and trabecular structures in the distal part of the femur. \u003cstrong\u003eB\u003c/strong\u003eMeasurement of Tb.BMD, Tb.BV/TV, Tb.Th, Tb.N, Tb.Sp, Ct.BMD, Ct.BV and Ct.Th in the femur. \u003cstrong\u003eC\u003c/strong\u003e Representative contour plots from flow cytometry for regulatory T-cells (Treg; CD4+ CD25+ FOXP3+); RANKL (CD254) producing Treg cells; and IFN-γ producing CD4+ cells from bone marrow. \u003cstrong\u003eD\u003c/strong\u003e Serum levels of RANKL. Data are expressed as the mean ± SD. Statistical significance was determined using one-way ANOVA for comparative analysis. *, P \u0026lt; 0.05 and **, P \u0026lt; 0.01 ***, P\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4539468/v1/089bae54f8e591158a75b431.png"},{"id":59269731,"identity":"be520d2e-33b2-48ba-8248-af214baa484b","added_by":"auto","created_at":"2024-06-28 12:19:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":608759,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModifications of lipid composition in thermogenic adipose tissue.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Representative images of H\u0026amp;E staining of BAT and iWAT sections obtained from 20-week-old mice housed at 22°C. Scale bar, 50 μm. \u003cstrong\u003eB\u003c/strong\u003eStatistical analysis of lipid area in BAT and lipid diameter in iWAT. \u003cstrong\u003eC,D\u003c/strong\u003e Representative western blots and band density measurements for PKA, p-PKA, HSL, p-HSL and ATGL in BAT of the control and BAT-specific mitochondrial dysfunction (BKO) mice. \u003cstrong\u003eE\u003c/strong\u003e Real-time PCR analysis of fatty acid oxidation related genes in the BAT of 20-week old control and BKO mice. \u003cstrong\u003eF\u003c/strong\u003e Quantification of serum free fatty acids. \u003cstrong\u003eG\u003c/strong\u003e Comparative analysis of BAT and serum metabolite levels in 20-week-old control and BKO mice. Statistical significance was analyzed by one-way ANOVA. *, P \u0026lt; 0.05 and **, P \u0026lt; 0.01 ***, P\u0026lt; 0.001 compared with the indicated group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4539468/v1/d395f2ae5d9d12b996e6e959.png"},{"id":59269730,"identity":"b6ba4f6b-7711-47d9-ad50-6991069dce9b","added_by":"auto","created_at":"2024-06-28 12:19:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":220449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment with palmitic acid increases the number of osteoclasts and promotes production of RANKL and IFN-γ in bone marrow T cells.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e TRAP staining of osteoclasts and the number of TRAP-positive osteoclasts.Scale bar: 200 μm. \u003cstrong\u003eB\u003c/strong\u003e RANKL producing cells in Foxp3+ Treg populations treated with or without palmitic acid. \u003cstrong\u003eC\u003c/strong\u003e IFN-γ producing cells within the CD4+ T-cell populations treated with or without palmitic acid. Statistical significance was analyzed by one-way ANOVA. *, P \u0026lt; 0.05 and **, P \u0026lt; 0.01 compared with the indicated group.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4539468/v1/127dc6ccbcd70bf3fc344461.png"},{"id":61596380,"identity":"dc910893-1314-41cc-a3a0-e928225306b8","added_by":"auto","created_at":"2024-08-01 17:26:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3295099,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4539468/v1/b5468d0e-12b4-4b9f-b621-18b717cccf44.pdf"},{"id":59269732,"identity":"16af5afa-594a-48d8-a3dc-46d59f331e8b","added_by":"auto","created_at":"2024-06-28 12:19:48","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1819376,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinfomation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4539468/v1/dec370bc772001bb99ed73c0.docx"}],"financialInterests":"","formattedTitle":"Brown fat-specific mitoribosomal function is crucial for preventing cold exposure-induced bone loss","fulltext":[{"header":"Introduction","content":"\u003cp\u003eExternal temperature, as an environmental factor, influences numerous physiological processes, necessitating continuous adaptation of organisms to variations in temperature. Temperature variations can impact the functioning of the nervous system[29], endocrine system[8], and musculoskeletal system.[1; 14] Exposure to warm conditions can influence developmental processes, as shown by increases in the growth of the femur[27] and promotion of a compact and structurally robust trabecular and cortical bone structures.[14] Heating one limb from an early age leads to the growth of extremities solely on the heated side.[28] Additionally, chondrocytes, when cultured in warm conditions, exhibit increased proliferation rates.[26] Recently, the link between ambient temperature and bone density has garnered significant scientific attention. However, the full impact of environmental temperature on bone balance remains unclear. Certain studies have suggested that low temperature may increase bone density.[1] Conversely, other research has indicated that low temperature may adversely affect bone volume (BV), exemplifying the contrasting impacts of low temperature on bone health.[14; 25] Exposure to cold temperatures is also known to influence the production and release of neurotransmitters involved in the regulation of skeletal metabolism and endocrine functions.[19; 33] Neuropeptide Y (NPY) has been reported to increase energy expenditure, upregulate uncoupling protein 1 (UCP-1) expression, and contribute to bone loss during cold exposure.[33] Activation of sympathetic nerves by low temperatures triggers β-adrenergic receptors, leading to the initiation of bone resorption processes by osteoclasts.[4; 10] Moreover, these activated sympathetic nerves indirectly impact bone mass by modulating the expression of bone morphogenetic protein 8b (BMP8b) and parathyroid hormone (PTH).[10; 11] Additionally, a recent paper reported that thermoneutral housing of mice mitigates bone loss induced by ovariectomy through the regulation of the gut microbiome environment.[6] Osteoporosis, a prevalent metabolic bone condition, is characterized by reduced bone density and the weakening of bone microarchitecture,[31] resulting in more fragile bones and an increased likelihood of fractures. The most common type of primary osteoporosis occurs as a result of post-menopausal estrogen deficiency,[23] and as such, it is exceedingly common in older women but can also occur in men. The impact of living temperature conditions during post-development and late adulthood, both in healthy individuals and those with osteoporotic conditions, on bone health, remodeling, and physiological processes remains uncertain.\u003c/p\u003e \u003cp\u003eMaintaining a constant body temperature in a changing temperature environment is critical for survival. When exposed to low temperatures, mammals increase oxidative metabolism for heat production. BAT is the main site of non-shivering thermogenesis in mammals. BAT activation is a complex physiological response to cold exposure, in which norepinephrine-mediated signaling promotes lipolysis, releasing free fatty acids and glycerol. These substrates fuel mitochondrial beta-oxidation and, via UCP-1, facilitate thermogenesis instead of ATP production,[30] thereby increasing energy expenditure and contributing to metabolic health. Brown adipocytes are characterized by their abundant mitochondria, which possess a high capacity for oxidation, and contain UCP-1 within their inner membranes.[24] BAT mitochondrial oxidative activity is increased by cold exposure.[18] UCP-1, which is unique to brown adipocytes, disrupts the link between the respiratory chain and oxidative phosphorylation, leading to increased oxidation rates and the conversion of metabolic energy into heat.[3] BAT volume has been reported to correlate with bone mass,[21] to influence positively bone mass, and to serve as a reliable indicator of the structural integrity of the femur.[2] A previous study reported that BAT-deficient mice show a marked reduction in bone formation.[22] Additionally, studies in human have identified brown adipose tissue as a significant independent factor influencing bone mass,[1; 20] and in mice, BAT has been shown to rescue bone loss induced by cold exposure at an early time point.[9] In the current study, we explored BAT function-related bone phenotypes using BAT-specific mitochondrial oxidative phosphorylation (OxPhos) deficient mice.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eMice\u003c/b\u003e \u003c/p\u003e \u003cp\u003e\u003cem\u003eUcp1-cre\u003c/em\u003e transgenic (Tg [\u003cem\u003eUcp-Cre\u003c/em\u003e]1Evdr) mice were purchased from Jackson Laboratory, and were backcrossed to C57BL/6J background mice. Floxed \u003cem\u003eCrif1\u003c/em\u003e (\u003cem\u003eCrif1\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e) mice were generated as previously described.[16] The mice in this study were kept in a specific pathogen-free environment at the Preclinical Research Center of Chungnam National University Hospital and were housed under standard conditions, which included a cycle of 12 h of light followed by 12 h of darkness, a stable room temperature of 22\u0026deg;C, and a relative humidity of between 40\u0026ndash;60%. Their diet consisted of Teklad global chow with 18% protein content (2918C, ENVIGO). Severe cold and thermoneutral housing conditions were performed at 14\u0026deg;C and 30\u0026deg;C, respectively, in a light and humidity-controlled chamber. All experimental procedures complied with the guidelines of Institutional Animal Care and Use Committees (IACUCs)\u003c/p\u003e \u003cp\u003e \u003cb\u003eMicro-CT analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMicro-CT was performed on vertebrae and long bones using SkyScan 1173 (SkyScan, Belgium) with 8 \u0026micro;m resolution. All bone morphometric parameters were calculated three-dimensionally with CTan and CTvox version 1.6, which was used to measure BV, total volume, BV/TV, bone surface, bone surface density, trabecular thickness, and trabecular separation. All bone micro-CT nomenclature followed the guidelines of the American Society for Bone and Mineral Research (ASBMR).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll Statistical analyses were conducted using GraphPad Prism software (version 9, Dotmatics, San Diego, USA). Data were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Unpaired Student's t-tests and one-way ANOVA followed by Scheffe's post-hoc test were used to determine statistical significance, with a p-value of less than 0.05 considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eLow temperature accelerates bone loss in mice\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTemperatures of 28\u0026ndash;33\u0026deg;C are regarded as thermoneutral housing conditions for mice based on studies of mouse metabolic rates at different temperatures. Typical room temperatures for housing mice can be considered mild cold conditions, whereas temperatures below 16\u0026deg;C are viewed as severe cold conditions, necessitating shivering thermogenesis.[12] To explore the effects of housing temperature on bone mass homeostasis, we employed micro-computed tomography (micro-CT) analysis to evaluate the bone structural features of SHAM and ovariectomized (OVX) mice, a commonly used primary osteoporosis model. After performing OVX and SHAM surgery on 8-week-old mice, the mice were maintained at 22\u0026deg;C, corresponding to standard room temperature housing conditions, and 30\u0026deg;C, corresponding to thermoneutral housing conditions, for 12 weeks, and the experiments were performed when the mice were 20 weeks old. Micro-CT analysis was conducted to assess cortical and trabecular bone architecture of SHAM and OVX mice housed under room temperature conditions and those housed under thermoneutral conditions. As expected, in trabecular bone, thermoneutral housing conditions significantly increased bone mineral density (BMD), BV, and trabecular number in OVX mice but not in SHAM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In cortical bone, housing temperature had no effect on these parameters in either SHAM or OVX mice (Supplementary Fig.\u0026nbsp;1A and 1B). Next, we examined the bone phenotypes under severe cold housing conditions (14\u0026deg;C). The SHAM mice housed under these conditions showed decreases in trabecular and cortical bone parameters compared with those housed under room temperature conditions. In OVX mice housed under severe cold conditions, trabecular BMD was significantly lower than in those housed under room temperature conditions (Supplementary Fig.\u0026nbsp;2A and 2B). Tartrate-resistant acid phosphatase (TRAP) staining revealed that the number of mature osteoclasts was significantly higher in OVX mice than in SHAM mice and significantly higher in OVX mice housed at 22\u0026deg;C than in those housed at 30\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). However, there was no significant difference in the serum level of P1NP, a bone formation marker, between mice housed at 22\u0026deg;C and those housed at 30\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Collectively, exposure to lower temperature appears to induce bone loss accompanied by an increase in the number of osteoclasts.\u003c/p\u003e\n\u003ch3\u003eTemperature-dependent modulation of bone marrow T-cells and BAT function\u003c/h3\u003e\n\u003cp\u003eNext, we investigated how housing temperature affects bone marrow immune cell phenotypes, which is an important factor in the initiation of bone resorption [32]. To address this issue, we examined the expression of osteoclastogenic cytokines in bone marrow cells from mice housed at 22\u0026deg;C and 30\u0026deg;C. We observed a decrease in the populations of CD4\u0026thinsp;+\u0026thinsp;CD25\u0026thinsp;+\u0026thinsp;Foxp3\u0026thinsp;+\u0026thinsp;regulatory T-cells (Tregs) in both OVX and SHAM mice at 22\u0026deg;C. Previous studies showed that Tregs produce RANKL, which is important for osteoclastogenesis and is linked to the bone damage observed in inflammatory arthritis [17]. We found that the expression of RANKL (CD254\u003csup\u003e+\u003c/sup\u003e) in Tregs was higher in OVX mice housed at 22\u0026deg;C than in those housed at 30\u0026deg;C. The expression of IFN-γ, a cytokine known to inhibit osteoclast differentiation, was lower in the CD4\u0026thinsp;+\u0026thinsp;cells of OVX mice housed at 22\u0026deg;C than in those housed at 30\u0026deg;C. The expression of IL-17A\u0026thinsp;+\u0026thinsp;was higher in CD4\u0026thinsp;+\u0026thinsp;cells of OVX mice than in those of SHAM mice, irrespective of housing temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These changes subsequently regulate the proliferation of osteoclasts. Additionally, we examined changes in BAT, an organ known for its temperature sensitivity. As reported previously, at lower temperatures, BAT exhibited significantly higher expression of UCP-1 and mitochondrial OxPhos complex subunits, including complex I (NDUFB8), II (SDHB), and IV (COX4) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Immunohistochemistry also revealed higher UCP-1 and succinate dehydrogenase (SDH) expression in BAT at lower temperatures in SHAM mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\n\u003ch3\u003eImpact of BAT removal on bone loss\u003c/h3\u003e\n\u003cp\u003eNext, we investigated whether BAT is directly involved in bone loss caused by cold exposure. At 8 weeks of age, mice were randomly assigned to undergo either a sham operation (SHAM mice) or surgical removal of interscapular BAT (BAT-deficient mice). Subsequently, the SHAM and BAT mice were housed at temperatures of either 22\u0026deg;C or 14\u0026deg;C for 12 weeks. The BAT-deficient mice housed at either of these temperatures did not show BAT regeneration at the end of the 12-week period. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The BAT mice housed at 22\u0026deg;C showed decreases in trabecular BMD, trabecular BV, and trabecular percent BV, and an increase in trabecular separation. Cortical BV was also decreased in the cortical bone region. The BAT mice housed at 14\u0026deg;C also tended to show decreases in bone parameters, but the decreases were not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), with the exception of trabecular BMD. These findings lend support to the hypothesis that surgical removal of BAT exacerbates bone loss induced by cold exposure.\u003c/p\u003e\n\u003ch3\u003eBAT-specific mitochondrial OxPhos dysfunctional mouse model\u003c/h3\u003e\n\u003cp\u003eMice with a tissue-specific deficiency of \u003cem\u003eCrif1\u003c/em\u003e (CR6-interacing factor 1), which encodes a protein of the large subunit of the mitochondrial ribosome, have markedly impaired mitoribosome-mediated translation [7; 15; 16]. In this study, to determine the impact of BAT mitochondrial OxPhos dysfunction on the bone, we generated BAT-specific \u003cem\u003eCrif1\u003c/em\u003e knockout (BKO) mice through selective disruption of \u003cem\u003eCrif1\u003c/em\u003e in brown adipocytes using the Cre-loxP system. \u003cem\u003eCrif1\u003c/em\u003e-floxed (\u003cem\u003eCrif1\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e) mice were bred with \u003cem\u003eUcp1-Cre\u003c/em\u003e transgenic mice, resulting in the deletion of exon 2 of the \u003cem\u003eCrif1\u003c/em\u003e gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). \u003cem\u003eCrif1\u003c/em\u003e deficiency resulted in reduced translation of CRIF1, OxPhos subunits, including complex I (NDUFB8), complex II (SDHB), complex III (UQCRC2), and complex IV (MTCO1), and decreased UCP-1 expression in mice housed under chronic cold (22\u0026deg;C) conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Blue native-PAGE (BN-PAGE) analysis of mitochondria isolated from BAT revealed reduced levels of native OxPhos complexes I and III (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Immunohistochemistry staining showed that BAT from the \u003cem\u003eCrif1\u003c/em\u003e-knockout had reduced succinate dehydrogenase (SDH) expression, confirming reduced mitochondrial oxidative phosphorylation in the OxPhos dysfunctional mice, and reduced UCP-1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Taken together, these findings imply that \u003cem\u003eCrif1\u003c/em\u003e deficiency in BAT sufficiently impairs OxPhos dysfunction in mice.\u003c/p\u003e\n\u003ch3\u003eMitochondrial oxidative phosphorylation dysfunction in BAT accelerates bone loss caused by chronic cold stress\u003c/h3\u003e\n\u003cp\u003eLoss of mitochondrial function in BAT is expected to result in multiple phenotypes upon cold exposure. Thus, we hypothesized that cold-induced activation of BAT would be compromised in BAT with mitochondrial oxidative phosphorylation dysfunction, leading to bone loss. To validate this hypothesis, we assessed the bone parameters of BKO mice at 20 weeks of age using micro-CT. In the trabecular region, the BKO mice housed at 22\u0026deg;C exhibited significant decreases in BMD, BV, percent BV, trabecular number and thickness, and an increase in trabecular separation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) compared with control mice housed under the same condition. Additionally, cortical BMD, BV, and thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) were lower in these mice than in the control mice. Furthermore, BKO mice maintained at 30\u0026deg;C for 12 weeks started to show reversal of bone loss in the trabecular region at 8 weeks of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Additionally, while there was no pronounced increase in BMD in cortical bone of BKO mice maintained at 30\u0026deg;C for 12 weeks, there was a discernible improvement in cortical BV and thickness (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). At a housing temperature of 22\u0026deg;C, BKO mice consistently displayed lower trabecular BMD and BV (Supplementary Fig.\u0026nbsp;3A and 3B) than control mice. Moreover, von Kossa staining showed decreased bone parameters not only in the femur but also in the vertebrae (Supplementary Fig.\u0026nbsp;3C). Taken together, these results indicate that BKO mice exhibit bone loss when exposed to chronic cold stress (22\u0026deg;C), but under thermoneutral conditions (30\u0026deg;C) have bone parameters similar to those of wild-type mice housed under the same conditions. This suggests that the activation of BAT mitochondria during cold exposure plays a pivotal role in maintaining bone mass.\u003c/p\u003e \u003cp\u003eNext, we investigated whether mitochondrial dysfunction in BAT affects the bone marrow immune environment, which is a critical determinant for the initiation of bone resorption, and further determined whether mitochondrial OxPhos dysfunction in BAT triggers osteoclastogenesis in bone marrow. To do this, we quantified various T-cell populations in the BM of control and BKO mice by flow cytometry analysis. We found that at 20 weeks of age, Treg populations were significantly larger in the bone marrow of BKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) than in that of control mice, Additionally, a subset of the Treg cell population expressing RANKL was also higher in BKO mice than in control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The serum level of RANKL was also increased in these mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Furthermore, in the CD4\u0026thinsp;+\u0026thinsp;T-cell population, IFN-γ expression, which is known to inhibit osteoclast differentiation, was lower in BKO mice than in control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Thus, at low temperatures (22\u0026deg;C) requiring thermogenesis, BKO mice with BAT mitochondrial dysfunction have a bone marrow immune environment that favors osteoclastogenesis. Under thermoneutral conditions (30\u0026deg;C), the expression of RANKL in Treg cells and IFN-γ in CD4\u0026thinsp;+\u0026thinsp;cells was lower than that in the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\n\u003ch3\u003eAlterations in lipid composition within thermogenic adipose tissue and corresponding metabolite variations in serum\u003c/h3\u003e\n\u003cp\u003eBrown fat tissue is a site of active lipid catabolism, resulting in the release fatty acids for energy utilization via lipolysis. These fatty acids fuel thermogenesis, a process in which energy is not converted into ATP but instead released as heat. This is critical for body temperature regulation, especially during cold stress. This thermogenic response is orchestrated by UCP-1, which is prevalent in the mitochondrial membrane of brown fat cells and disrupts the typical pathway of oxidative phosphorylation.[5] In this study, we observed a decrease in the lipid area within BAT when the mice were housed at low temperatures (22\u0026deg;C and 14\u0026deg;C) (Supplementary Fig.\u0026nbsp;4A and 4B), Furthermore, Western blot analysis confirmed that cold temperature housing conditions activate lipolysis (Supplementary Fig.\u0026nbsp;4C and 4D), suggesting that BAT may counteract low temperatures by inducing thermogenesis using the energy derived from lipolysis. Next, we hypothesized that BAT in BKO mice would exhibit impaired lipolysis and fatty acid oxidation. To test this hypothesis, we examined the BAT phenotype of BKO mice. The BKO mice had larger lipid areas in BAT and larger lipid droplet sizes in inguinal white adipose tissue (iWAT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Additionally, Western blot analysis revealed reduced lipolysis in BAT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD) and RT-PCR revealed reduced expression of genes controlling fatty acid oxidation (\u003cem\u003eCpt1a, Cpt1b, Acadm, Ppara\u003c/em\u003e, and \u003cem\u003ePgc1a\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Furthermore, BKO mice had elevated serum levels of free fatty acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), which may be attributed to decreased lipolysis and fatty acid oxidation in these mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, the increased metabolites in the BAT and serum of BKO mice were predominantly fatty acids, with a notable increase in long-chain fatty acid levels in serum. Collectively, dysfunctional BAT mitochondria impair BAT fatty acid oxidation, leading to elevated long-chain fatty acids in the bloodstream.\u003c/p\u003e\n\u003ch3\u003eLong-chain fatty acids activate osteoclasts and promote T-cell osteoclastogenic transformation\u003c/h3\u003e\n\u003cp\u003eReductions in lipolysis and fatty acid oxidation in BAT increase serum free fatty acids levels and decrease bone mass in BKO mice, suggesting that serum free fatty acids may directly or indirectly influence bone marrow cells. To determine the effects of fatty acids on bone marrow cells, we treated various types of bone marrow cells with different fatty acids. First, we treated bone marrow-derived osteoclasts with short-chain fatty acids (propionic acid) and long-chain fatty acids (palmitic acid). TRAP staining revealed no changes in the number of TRAP-positive osteoclasts after treatment with propionic acid compared with treatment with vehicle; however, a marked increase in the number of TRAP-positive osteoclasts was observed when the osteoclasts were treated with palmitic acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Next, we examined for potential changes in bone marrow immune cell phenotypes following treatment with long-chain fatty acids by performing flow cytometry of bone marrow immune cells treated with palmitic acid for 24 h. Upon treatment with palmitic acid, the number of RANKL-expressing Tregs increased dose-dependently with the concentration of palmitic acid (50 \u0026micro;M and 100 \u0026micro;M), whereas the number of IFN-γ expressing CD4\u0026thinsp;+\u0026thinsp;T cells significantly decreased compared with the vehicle-treated control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Taken together, these results suggest that, among the metabolites altered by the reduced lipolysis or fatty acid oxidation in BAT, long-chain fatty acids (palmitic acid) directly or indirectly regulate osteoclast differentiation, potentially leading to bone loss.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe relationship between ambient temperature and bone physiology is a complex and multifaceted one, with a growing body of evidence suggesting that environmental factors play a significant role in the regulation of bone metabolism. This study reveals that temperature is a critical determinant of bone remodeling, which has profound implications for our understanding of bone health and disease management.\u003c/p\u003e \u003cp\u003eThe impact of temperature on bone physiology can also be seen through the lens of thermoregulatory metabolism. Mild cold stress, common in standard mouse housing conditions, is known to affect tumor growth rates, CD8\u0026thinsp;+\u0026thinsp;T-cell and dendritic cell function, and the activity of immunosuppressive cells.[13] This underscores the importance of considering environmental factors when studying bone physiology and its interactions with the immune system. Our study also explored the temperature-dependent changes in bone marrow T-cell populations, shedding light on the immune-mediated mechanisms that may contribute to bone remodeling. We observed an increase in Tregs, which are known to suppress osteoclastogenesis, at thermoneutral temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggests that a warmer ambient temperature may protect against bone loss by modulating the immune environment within bone marrow. Conversely, at low temperatures, we noted an increase in the pro-resorptive T-cell populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which could contribute to the increased bone resorption observed at these temperatures. The results potentially highlight the importance of immune system interactions in mediating the effect of temperature on bone density.\u003c/p\u003e \u003cp\u003eAn intriguing aspect of our study is the relationship between BAT activity and bone mass. BAT is known to play an important role in thermogenesis, the process via which heat is produced in organisms. Brown adipocytes are rich in mitochondria, which have a high oxidative capacity and contain UCP-1 in their inner membrane,[24] which is particularly active during cold exposure [18]. Numerous studies have reported a positive relationship between BAT activity and bone mass [1; 2; 20\u0026ndash;22]. However, research into the mechanisms underlying the relationship between BAT and bone mass remains limited. Our data suggest that BAT activity correlates positively with bone mass, indicating that BAT protects against bone loss. This is further corroborated by the observation that mice lacking BAT have a reduced bone mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The thermogenic function of BAT, primarily driven by UCP-1 and mitochondrial oxidative phosphorylation, may be a key part of this protective mechanism. This is not only due to the thermogenerative properties of BAT but also to the metabolic activity associated with thermogenesis, which appears to have a systemic effect on bone tissue. Furthermore, our research suggests that BAT mitochondrial functions are crucial for bone health, particularly under cold stress conditions. Mice with compromised BAT mitochondrial functions exhibited accelerated bone loss when exposed to the cold, but not when exposed to thermoneutral conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This underscores the importance of BAT mitochondrial integrity for the preservation of bone mass during environmental stress.\u003c/p\u003e \u003cp\u003eThe metabolic mechanisms that mediate the effects of temperature variations on bone physiology are multifaceted. Our research suggests that lipolysis and β-oxidation in BAT play a pivotal role in modulating the levels of systemic metabolites. Lipolysis, a critical process for cold-induced thermogenesis, is intricately connected to bone metabolism through alterations in metabolite profiles. We found that reduced mitochondrial function in BKO mice was linked to decreases in lipolysis and fatty acid oxidation, leading to an increase in fatty acid-based metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Furthermore, among the increased metabolites, long-chain fatty acids not only increased the differentiation of osteoclasts as evidenced by increased TRAP staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), but also induced changes in cytokine expression in T cells, such as RANKL expression in Tregs and IFN-γ expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), thereby potentially exerting indirect control over osteoclast regulation. However, our study is not without its limitations. Although we observed that housing temperature affected bone mass and BAT confers protection against bone loss induced by cold exposure, the mechanisms responsible for cold-induced bone loss were not clearly identified in this study.\u003c/p\u003e \u003cp\u003eIn conclusion, chronic cold exposure is associated with alterations in immune cells and is linked to bone loss. BAT plays a crucial role in protecting against bone loss induced by cold exposure, and this protective effect of BAT is linked to mitochondrial fatty acid oxidation and lipolysis-driven thermogenesis, as well as the consumption of long-chain fatty acids. Mitochondrial dysfunction in BAT is known to lead to an increase in systemic long-chain fatty acid levels, Our in vitro experiments support these findings, indicating that long-chain fatty acids can directly and indirectly upregulate osteoclasts. in turn promoting osteoclastogenic T-cell activity and bone loss.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJT and HSY designed the study and wrote the manuscript. JT, JSM, HYL, and DS performed in vitro and in vivo experiments. HTN, TLN, and HJJ helped with data collection and assembly. MS and JHL provided helpful suggestions for project design and comments for the improvement of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from the Korea Health Technology R\u0026amp;D Project, through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health \u0026amp; Welfare, Republic of Korea (grant number: HR22C1734 and HI23C153400). HSY was supported by the Basic Science Research Program, through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning, Korea (NRF-2023R1A2C3006220 and NRF-2021R1A5A8029876).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information files. All primary data will be shared by the lead contact upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthicla approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the animal experiments were approved by the Institutional Animal Care and Use Committee of Chungnam National University School of Medicine (CNUH-2023-IA0036-00)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors provided consent for publication\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBredella MA, Fazeli PK, Freedman LM, Calder G, Lee H, Rosen CJ, Klibanski A (2012) Young women with cold-activated brown adipose tissue have higher bone mineral density and lower Pref-1 than women without brown adipose tissue: a study in women with anorexia nervosa, women recovered from anorexia nervosa, and normal-weight women. 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Eur J Rheumatol 4(1):46-56 https://doi.org/10.5152/eurjrheum.2016.048\u003c/li\u003e\n\u003cli\u003eTian J, Chung HK, Moon JS, Nga HT, Lee HY, Kim JT, Chang JY, Kang SG, Ryu D, Che X, Choi JY, Tsukasaki M, Sasako T, Lee SH, Shong M, Yi HS (2022) Skeletal muscle mitoribosomal defects are linked to low bone mass caused by bone marrow inflammation in male mice. J Cachexia Sarcopenia Muscle 13(3):1785-1799 https://doi.org/10.1002/jcsm.12975\u003c/li\u003e\n\u003cli\u003eWee NKY, Nguyen AD, Enriquez RF, Zhang L, Herzog H, Baldock PA (2020) Neuropeptide Y Regulation of Energy Partitioning and Bone Mass During Cold Exposure. Calcif Tissue Int 107(5):510-523 https://doi.org/10.1007/s00223-020-00745-9\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cold exposure, bone loss, brown adipose tissue, mitochondria","lastPublishedDoi":"10.21203/rs.3.rs-4539468/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4539468/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigates the relationship between ambient temperature, brown adipose tissue (BAT) function, and bone metabolism, revealing how cold exposure and BAT mitochondrial activity influence bone health. Utilizing ovariectomized (OVX) mice to model primary osteoporosis and BAT-specific mitochondrial dysfunction (BKO) mice, we explored the effects of housing temperature on bone density, immune modulation in bone marrow, and the protective role of BAT against bone loss.\u003c/p\u003e \u003cp\u003eThe results showed that cold exposure universally decreases bone mass, increases osteoclastogenesis, and shifts bone marrow T-cell populations, suggesting a key role of the immune system in bone remodeling under cold stress. Crucially, the thermogenic function of BAT, underpinned by mitochondrial oxidative phosphorylation, protected against bone loss. Impairments in BAT function, either through surgical removal or mitochondrial dysfunction, exacerbated bone loss in cold environments, highlighting the importance of BAT metabolic activity for bone health.\u003c/p\u003e \u003cp\u003eAdditionally, we found that cold-induced alterations in BAT function led to systemic metabolic changes, including increased long-chain fatty acid levels, which directly and indirectly affected osteoclast differentiation and activity. These findings point to a systemic mechanism via which environmental temperature and BAT metabolism are interconnected with bone physiology, offering new insights into the metabolic and environmental determinants of bone health.\u003c/p\u003e \u003cp\u003eIn summary, our study underscores the complex interactions between ambient temperature, BAT function, and bone health, suggesting that novel bone disease therapies could be developed by targeting metabolic and environmental factors. Further research in these pathways may provide innovative approaches for managing bone health amid changing environmental conditions and metabolic dysfunctions.\u003c/p\u003e","manuscriptTitle":"Brown fat-specific mitoribosomal function is crucial for preventing cold exposure-induced bone loss","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-28 12:19:43","doi":"10.21203/rs.3.rs-4539468/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2024-05-22T23:04:34+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-04-28T13:50:28+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-28T13:24:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2024-04-18T02:09:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b9956f1a-e26e-4257-8eca-53761b7e7c16","owner":[],"postedDate":"June 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T17:10:16+00:00","versionOfRecord":{"articleIdentity":"rs-4539468","link":"https://doi.org/10.1007/s00018-024-05347-4","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2024-07-27 16:15:59","publishedOnDateReadable":"July 27th, 2024"},"versionCreatedAt":"2024-06-28 12:19:43","video":"","vorDoi":"10.1007/s00018-024-05347-4","vorDoiUrl":"https://doi.org/10.1007/s00018-024-05347-4","workflowStages":[]},"version":"v1","identity":"rs-4539468","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4539468","identity":"rs-4539468","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}