Supplementary Material for: Diet Stimulated Marrow Adiposity Fails to Worsen Early, Age-Related Bone Loss

Introduction
Longitudinal effect of diet-induced obesity on bone is uncertain. Prior work showed both no effect versus a decrement in bone density or quality when obesity begins prior to skeletal maturity. We aimed to quantify long-term effects of obesity on bone and bone marrow adipose tissue (BMAT) in adulthood.
Methods
Skeletally mature, female C57BL/6 mice (n=70) aged 12-weeks were randomly allocated to low-fat (LFD; 10%kcal fat; n=30) or high-fat (HFD; 60%kcal fat; n=30) diet, with analyses at 12,15,18 and 24-weeks (n=10/group). Tibial microarchitecture was analyzed by µCT, and volumetric BMAT was quantified via 9.4T MRI/advanced-image-analysis. Histomorphometry of adipocytes and osteoclasts, and qPCR were performed.
Results
Body weight and visceral white adipose tissue accumulated in response to HFD started in adulthood. Trabecular bone parameters declined with advancing experimental age. BV/TV declined 22% in LFD (p=0.0001) and 17% in HFD (p=0.0022) by 24-weeks. HFD failed to appreciably alter BV/TV and had negligible impact on other microarchitecture parameters. Both dietary intervention and age accounted for variance in BMAT, with regional differences: distal femoral BMAT was more responsive to diet, while proximal femoral BMAT was more attenuated by age. BMAT increased 60% in the distal metaphysis in HFD at 18 and 24 weeks (p=0.0011). BMAT in the proximal femoral diaphysis, unchanged by diet, decreased 45% due to age (p=0.0002). Marrow adipocyte size via histomorphometry supported MRI quantification. Osteoclast number did not differ between groups. Tibial qPCR showed attenuation of some adipose, metabolism, and bone genes. A regulator of fatty-acid β-oxidation, Cytochrome C (CYCS), was 500% more abundant in HFD-bone (p<0.0001; diet effect). CYCS also increased due to age, but to a lesser extent. HFD mildly increased OCN, TRAP, and SOST.
Conclusions
Long-term high fat feeding after skeletal maturity, despite upregulation of visceral adiposity, body weight, and BMAT, failed to attenuate bone microarchitecture. In adulthood, we found aging to be a more potent regulator of microarchitecture than diet-induced obesity.

引言
饮食诱导肥胖对骨骼的纵向影响目前尚未明确。既往研究表明，若在骨骼成熟前诱导肥胖，可观察到骨密度或骨质量无显著变化，亦或出现下降。本研究旨在量化成年期肥胖对骨骼及骨髓脂肪组织（bone marrow adipose tissue, BMAT）的长期影响。

方法
选取12周龄、骨骼发育成熟的雌性C57BL/6小鼠共70只，随机分为低脂饲料组（LFD，热量中10%来自脂肪，n=30）与高脂饲料组（HFD，热量中60%来自脂肪，n=30），分别于实验第12、15、18、24周进行检测，每组每次检测10只小鼠。采用显微CT（µCT）分析胫骨骨微结构，通过9.4T磁共振成像（MRI）结合先进图像分析技术定量检测体积骨髓脂肪组织。同时开展脂肪细胞与破骨细胞的组织形态计量学检测，以及实时定量聚合酶链反应（qPCR）实验。

结果
成年小鼠经高脂饲料干预后，体重与内脏白色脂肪组织均出现蓄积。骨小梁参数随实验周期延长逐渐降低。至实验第24周时，骨体积分数（BV/TV, bone volume/tissue volume）在低脂饲料组下降22%（p=0.0001），高脂饲料组下降17%（p=0.0022）。高脂饲料组未显著改变骨体积分数，对其余骨微结构参数亦无明显影响。
饮食干预与年龄均可影响骨髓脂肪组织的水平，且存在区域差异：股骨远端骨髓脂肪组织对饮食干预的响应更为显著，而股骨近端骨髓脂肪组织则更多受年龄因素调控。实验第18、24周时，高脂饲料组股骨远端干骺端的骨髓脂肪组织水平升高60%（p=0.0011）。股骨近端骨干的骨髓脂肪组织不受饮食干预影响，但随年龄增长下降45%（p=0.0002）。组织形态计量学检测的骨髓脂肪细胞大小结果，验证了磁共振成像的定量分析结果。两组间破骨细胞数量无显著差异。
胫骨组织的实时定量PCR检测显示，部分脂肪代谢、能量代谢及骨骼相关基因的表达出现下调。作为脂肪酸β氧化调控因子的细胞色素C（Cytochrome C, CYCS）在高脂饲料组骨组织中的表达量升高500%（p<0.0001，饮食干预效应）。细胞色素C的表达量亦随年龄增长而升高，但升高幅度较小。高脂饲料组的骨钙素（OCN, osteocalcin）、抗酒石酸酸性磷酸酶（TRAP, tartrate-resistant acid phosphatase）及骨硬化蛋白（SOST, sclerostin）的表达水平轻度升高。

结论
骨骼成熟后长期给予高脂饲料，尽管可导致内脏脂肪蓄积、体重增加及骨髓脂肪组织水平升高，但并未削弱骨微结构。本研究发现，在成年阶段，年龄因素对骨微结构的调控作用强于饮食诱导的肥胖。