成骨细胞beta-catenin基因缺失致内脏脂肪减少的机制研究

The prevalence of osteoporosis and obesity goes higher in recent years, and some results have shown that these two diseases are related with each other. Recent studies have highlighted role of osteoblasts to ensure this energy supply through osteoblast-secreted osteocalcin dependent and independent mechanisms. Mice with induced osteoblast deficiency displayed hypoinsulinemia, hyperglycemia, glucose intolerance, decreased gonadal fat, and increased energy expenditure. Treatment of these mice with osteocalcin could normalize glucose and insulin levels； however, it did not have any effect on restoring body fat. Therefore, several molecular mechanisms of involved in osteoblast regulation of energy homeostasis and fat accumulation remain to be elucidated. In this study, we aim to study the role of beta-catenin pathway in osteoblast on regulating visceral fat accumulation. To this end, we have generated mice in which beta-catenin could be deleted in pre-osteoblasts in inducible manner (OB-beta-catenin KO). Preliminary data show that conditional disruption of beta-catenin in pre-osteoblasts resulted in markedly decreased visceral fat and lean phenotype along with severe osteopenia. The dramatic decrease in numbers of osteoblast indicates decreased osteocalcin synthesis. However, in contrast to the obesity observed in osteocalcin deficient mice, the decrease in body fat in OB-beta-catenin KO mice suggests osteocalcin independent mechanism of action. In this study, we hypothesize that beta-catenin signaling in osteoblast regulates whole body fat accumulation via independent mechanism. First, we will measure energy metabolism and histology of white and brown fat of this mice model,then mark the beta-catenin KO osteoblsts with GFP in vivo to assure the isolation of these gene KO osteoblasts by flow cytometry. Culture the isolated cells and get the condition media,then the precursor cells of white and brown fat are cultured under the condition media to detect how their proliferation and differentiation are regulated. After normalized the osteocalcin level in condition media, the secretome of beta-catenin KO osteoblasts will be evaluated by 2-dimensional electrophoresis (2-DE), then the proteins with different expression level to the control osteoblasts will be selected. These proteins will be analyzed to check their functions and to tell their contributions to the proliferation and differention of precursor cells of white and brown fat. These studies will help us further understand the correlation between bone and fat deposition in visceral fat accumulation. Identification of the novel molecular mechanisms may offer new therapies for obesity.

骨质疏松和肥胖的患病人数逐年增多，研究发现骨骼与脂肪组织呈闭环状相互调节，成骨细胞可通过骨钙素依赖和非依赖途径调节脂代谢，而后一途径的机理尚不清楚。我们前期研究显示，成骨细胞β-catenin基因敲除小鼠成骨细胞数量和功能降低，伴有内脏脂肪明显减少，提示该模式小鼠中成骨细胞对脂肪生成的调节不依赖骨钙素，本研究将进一步探讨其发生机制。首先检测该模式小鼠能量代谢和白色、棕色脂肪的组织学特征，然后用GFP体内标记、流式细胞术分选获得基因敲除的成骨细胞，取该细胞的培养上清液，观察其对白色、棕色脂肪前体细胞增殖和分化的影响。为明确可影响脂肪形成的因子，纠正骨钙素至对照水平后，检测基因敲除成骨细胞培养上清液中分泌蛋白组的变化，对差异蛋白进行分析和鉴定，探讨β-catenin基因敲除成骨细胞致内脏脂肪减少的非骨钙素依赖途径。本研究旨在为发现可调节脂肪蓄积的骨源性蛋白提供线索，期望为肥胖的治疗提供新思路。

肥胖、糖尿病和骨质疏松的发病率逐年增高，已经对公众健康造成了较大的威胁，理解这些疾病的发病机制将有助于开发新的治疗方法、提高治疗效果。参与骨形成的成骨细胞系细胞已被证实可影响全身能量代谢，本研究旨在探讨成骨细胞系中wnt/β-catenin通路对全身能量代谢的影响。.本研究应用osterix-cre转基因小鼠、β-catenin flox/flox小鼠和Osteoprotegerin (OPG)过表达小鼠模型，获得成骨细胞中时间和组织特异性β-catenin基因敲除小鼠及同时过表达Osteoprotegerin的基因敲除小鼠，用β-catenin flox/flox小鼠作为对照小鼠，监测血糖、葡萄糖耐量、胰岛素耐量、血清胰岛素、胰岛细胞增殖情况等糖代谢指标，定量CT测定小鼠腹内和皮下脂肪组织和脂肪组织形态学指标，间接代谢笼测定小鼠的代谢指标，应用micro-CT测定小鼠骨骼定量等。由于骨钙素参与能量代谢，本研究外源补充骨钙素后观察β-catenin基因缺失小鼠的能量代谢变化情况。.研究结果显示，成骨细胞中wnt/β-catenin通路可调节全身能量代谢，包括糖代谢、脂肪蓄积和能量消耗。成骨细胞中β-catenin基因缺失小鼠具有低骨量、高血糖、低胰岛素、脂肪蓄积减少及高能量消耗的表型。外源补充骨钙素后可通过改善胰岛素分泌纠正β-catenin基因缺失小鼠的糖代谢异常，但不能改善该小鼠的脂肪蓄积减少和高能量代谢表型。通过与Osteoprotegerin过表达小鼠杂交后，过表达Osteoprotegerin可纠正β-catenin基因缺失小鼠的低骨量、减少的脂肪蓄积和增加的能量代谢。进一步针对机制的研究发现，与能量代谢相关的激素（甲状腺激素、肾上腺素和生长激素）水平在这些小鼠模型中并未改变，且这些小鼠模型并未发生脂肪组织的棕色化改变。.我们的研究结果表明，骨骼对能量代谢和脂肪蓄积的调节不仅仅通过骨钙素发挥，骨骼组织尚通过其他方式影响小鼠的脂肪蓄积和能量代谢。