RBP2 Biology and Pathobiology

摘要 这个项目的目标一直是，并将继续了解我们的生物化学基础 观察到视黄醇结合蛋白2(RBP2)在维持体重方面具有意想不到的作用， 对葡萄糖挑战的反应正常，空腹肝脏甘油三酯水平正常。我们发现，当 6-7个月大的Rbp2基因缺陷(Rbp2-/-)小鼠仅在对照组饮食中获得更多 体重作为白色脂肪组织(Wat)，对葡萄糖挑战的反应明显较差，并拥有 与匹配的野生型(WT)产仔对照相比，肝脏脂肪显著增加。这些表型完全是 由更年轻的55天大的Rbp2-/-和匹配的WT小鼠概括，喂食高脂肪食物6-7周。 我们发表的工作证实，RBP2和视黄醇结合蛋白4(RBP4)在 肠道内的肠内分泌细胞(EECs)，包括葡萄糖依赖的促胰岛素多肽(GIP) 和表达胰升糖素样肽1(GLP-1)的细胞。Rbp2-/-小鼠表现出循环GIP水平升高 对口腔脂肪挑战的反应。我们进一步证明了RBP2结合了长链不饱和的2- 单酰甘油(2-MAGS)与视黄醇具有同样高的亲和力，因此在生理上是一种 相关的2-MAG结合蛋白以及维甲酸结合蛋白。我们发现Rbp4-/-小鼠有 空腹和口服脂肪刺激后循环GIP浓度显著降低 WT控件。RBP4定位于EEC分泌颗粒，并与GIP共同分泌。 培养富含EEC的原代小鼠肠道细胞。由于GIP水平升高与 在人类和动物模型中的体重和脂肪质量，我们假设这对 Rbp2-/-小鼠的代谢表型。我们提出了探索RBP行动的三个具体目标， RBP4、维甲酸和2-MAG信号在调节EEC活性以及GIP和GLP-1的合成/分泌中的作用。 特定目标1将评估RBP2、全反式维甲酸(ATRA)和ATRA-RAR的参与 信号转导，以及RBP4在调节EEC反应中的作用。这需要进行体内和体外研究。 具体目标2将评估2-MAG和负责2-MAG合成的酶的参与。 2-MAG降解(单甘油脂肪酶)和α/β水解酶结构域 6和12(ABDH6和ADBH12)和2-MAG作用(细胞表面大麻素受体1和2(CB1和 Cb2)和GPR119受体)在调节K细胞对食物营养供应的反应中起重要作用。这些研究将 使用与具体目标1相同的方法。 具体目标3将提供更多关于RBP2是否在GLP-1中起直接作用的理解 在小肠和结肠中的合成/分泌。尽管我们已经报告了 L细胞中的Rbp2蛋白，与GIP和K细胞不同，我们还没有从 RBP2在生物学中可能扮演什么角色的观点。

ABSTRACT The goal of this project has been and remains to gain understanding of the biochemical basis for our observation that retinol-binding protein 2 (RBP2) has an unsuspected role in the maintenance of body weight, normal responses to a glucose challenge, and normal fasting hepatic triglyceride levels. We found that when maintained solely on a control chow diet, 6-7-month-old Rbp2-deficient (Rbp2-/-) mice accrue significantly more body weight as white adipose tissue (WAT), respond significantly less well to a glucose challenge, and possess significantly more hepatic fat than matched wild type (WT) littermate controls. These phenotypes were fully recapitulated by younger 55-day old Rbp2-/- and matched WT mice fed a high fat diet for 6-7 weeks. Our published work establishes that both RBP2 and retinol-binding protein 4 (RBP4) are expressed in enteroendocrine cells (EECs) within the gut, including by glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) expressing cells. Rbp2-/- mice show elevated circulating GIP levels in response to an oral fat challenge. We further showed that RBP2 binds long-chain unsaturated 2- monoacylglycerol (2-MAGs) with an equally high affinity as it does retinol and is consequently a physiologically relevant 2-MAG-binding protein as well as a retinoid-binding protein. We identified that Rbp4-/- mice have significantly diminished circulating GIP concentrations both fasting and after an oral fat challenge compared to WT controls. RBP4 localizes to EEC secretory granules and is co-secreted along with GIP upon stimulation of cultured EEC-enriched primary mouse intestinal cells. Since elevated GIP levels are associated with increased body weight and adipose mass in both humans and animal models, we hypothesize that this contributes to the metabolic phenotypes seen for Rbp2-/- mice. We are proposing 3 Specific Aims exploring the actions of RBP2, RBP4, retinoid- and 2-MAG signaling in regulating EEC activities and synthesis/secretion of GIP and GLP-1. Specific Aim 1 will assess the involvement of RBP2, all-trans-retinoic acid (ATRA) and ATRA-RAR signaling, and RBP4 in modulating EEC responses. This entails both in vivo and in vitro studies. Specific Aim 2 will assess the involvement of 2-MAGs and enzymes responsible for 2-MAG synthesis (diacylglycerol lipase (DAGL)), 2-MAG degradation (monoacylglycerol lipase (MAGL) and α/β hydrolase domain 6 and 12 (ABDH6 and ADBH12)) and 2-MAG actions (the cell surface cannabinoid receptors 1 and 2 (Cb1 and Cb2) and GPR119 receptors) in modulating K-cell responses to dietary nutrient availability. These studies will make use of the same methodologies employed in Specific Aim 1. Specific Aim 3 will provide greater understanding of whether RBP2 has a direct role in GLP-1 synthesis/secretion in both the small intestine and in the colon. Although we have reported the presence of RBP2 protein in L-cells, unlike GIP and K-cells, we have not systematically studied GLP-1 or L-cell biology from the perspective of what role RBP2 may have in this biology.