Son Yeonho, Choi Cheoljun, Saha Abhirup, Park Ji-Hyun, Im Hyeonyeong, Cho Yoon Keun, Seong Je Kyung, Burl Rayanne B, Rondini Elizabeth A, Granneman James G, Lee Yun-Hee
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Republic of Korea.
Korea Mouse Phenotyping Center (KMPC), and Laboratory of Developmental Biology and Genomics, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea.
Metabolism. 2022 May;130:155159. doi: 10.1016/j.metabol.2022.155159. Epub 2022 Feb 9.
The mobilization and catabolism of lipid energy is a central function of adipocytes that is under the control of the β-adrenergic signaling pathway, and defects in β-adrenergic signaling in adipocytes have been linked to obesity and obesity-related metabolic diseases. Receptor expression-enhancing proteins (REEPs) are endoplasmic reticulum (ER) proteins that play critical roles in subcellular targeting of receptor signaling complexes. Examination of gene expression profiles indicates that, among REEPs expressed in adipocytes, REEP6 expression is uniquely upregulated by sympathetic nervous system activation, suggesting involvement in regulating adrenergic signal transduction.
The aim of this study was to assess the contribution of REEP6 to the thermogenic activation of adipocytes and characterize the metabolic consequences of REEP6 deficiency in vivo.
Expression levels of Reep6 in adipose tissue were examined by using public transcriptomic data and validated by Western blot and qPCR analyses. Adipocyte-specific regulatory roles of REEP6 were investigated in vitro in C3H10T1/2 adipocytes and in primary adipocytes obtained from REEP6 KO mice. Effects of in vivo REEP6 deficiency on energy expenditure were measured by indirect calorimetry. Mitochondrial content in adipose tissue was accessed by immunoblot, mitochondrial DNA analysis, and confocal and electron microscopy. Effects of REEP6 KO on obesity-induced metabolic dysfunction were tested in a high-fat diet-induced obesity mouse model by glucose tolerance test, Western blot, and histological analyses.
REEP6 expression is highly enriched in murine adipocytes and is sharply upregulated upon adipocyte differentiation and by cold exposure. Inactivation of REEP6 in mice increased adiposity, and reduced energy expenditure and cold tolerance. REEP6 KO severely reduced protein kinase A-mediated signaling in BAT and greatly reduced mitochondrial mass. The effect of REEP6 inactivation on diminished β-adrenergic signaling was reproduced in cultured adipocytes, indicating that this effect is cell-autonomous. REEP6 KO also suppressed expression of adenylate cyclase 3 (Adcy3) in brown adipose tissue and knockdown of REEP6 in adipocytes reduced targeting of ADCY3 to the plasma membrane. Lastly, REEP6 KO exacerbated high-fat diet-induced insulin resistance and inflammation in adipose tissue.
This study indicates that REEP6 plays an important role in β-adrenergic signal transduction in adipocytes involving the expression and trafficking of Adcy3. Genetic inactivation of REEP6 reduces energy expenditure, increases adiposity, and the susceptibility to obesity-related metabolic dysfunction.
脂质能量的动员和分解代谢是脂肪细胞的核心功能，受β-肾上腺素能信号通路控制，脂肪细胞中β-肾上腺素能信号缺陷与肥胖及肥胖相关代谢疾病有关。受体表达增强蛋白（REEPs）是内质网（ER）蛋白，在受体信号复合物的亚细胞靶向中起关键作用。基因表达谱分析表明，在脂肪细胞中表达的REEPs中，REEP6的表达在交感神经系统激活时独特地上调，提示其参与调节肾上腺素能信号转导。
本研究旨在评估REEP6对脂肪细胞产热激活的作用，并在体内表征REEP6缺乏的代谢后果。
利用公开的转录组数据检测脂肪组织中Reep6的表达水平，并通过蛋白质免疫印迹和定量聚合酶链反应分析进行验证。在C3H10T1/2脂肪细胞和从REEP6基因敲除（KO）小鼠获得的原代脂肪细胞中体外研究REEP6的脂肪细胞特异性调节作用。通过间接测热法测量体内REEP6缺乏对能量消耗的影响。通过免疫印迹、线粒体DNA分析以及共聚焦和电子显微镜检测脂肪组织中的线粒体含量。在高脂饮食诱导的肥胖小鼠模型中，通过葡萄糖耐量试验、蛋白质免疫印迹和组织学分析测试REEP6基因敲除对肥胖诱导的代谢功能障碍的影响。
REEP6在小鼠脂肪细胞中高度富集，在脂肪细胞分化和冷暴露后急剧上调。小鼠中REEP6失活增加了肥胖程度，降低了能量消耗和耐寒性。REEP6基因敲除严重降低了棕色脂肪组织中蛋白激酶A介导的信号传导，并大大减少了线粒体质量。在培养的脂肪细胞中重现了REEP6失活对β-肾上腺素能信号减弱的影响，表明这种影响是细胞自主的。REEP6基因敲除还抑制了棕色脂肪组织中腺苷酸环化酶3（Adcy3）的表达，脂肪细胞中REEP6的敲低减少了ADCY3向质膜的靶向定位。最后，REEP6基因敲除加剧了高脂饮食诱导的胰岛素抵抗和脂肪组织炎症。
本研究表明REEP6在脂肪细胞β-肾上腺素能信号转导中起重要作用，涉及Adcy3的表达和转运。REEP6的基因失活降低了能量消耗，增加了肥胖程度以及对肥胖相关代谢功能障碍的易感性。
