Flanders Institute for Biotechnology, Department of Medical Protein Research, Ghent University, Faculty of Medicine and Health Sciences, A. Baertsoenkaai 3, 9000 Ghent, Belgium.
Biochem J. 2012 Jan 1;441(1):425-34. doi: 10.1042/BJ20110438.
The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. Accumulating evidence suggests that leptin plays a role in human pathologies, such as autoimmune diseases and cancer, thus providing a rationale for the development of leptin antagonists. In the present study, we generated and evaluated a panel of neutralizing nanobodies targeting the LR (leptin receptor). A nanobody comprises the variable domain of the naturally occurring single-chain antibodies found in members of the Camelidae family. We identified three classes of neutralizing nanobodies targeting different LR subdomains: i.e. the CRH2 (cytokine receptor homology 2), Ig-like and FNIII (fibronectin type III) domains. Only nanobodies directed against the CRH2 domain inhibited leptin binding. We could show that a nanobody that targets the Ig-like domain potently interfered with leptin-dependent regulation of hypothalamic NPY (neuropeptide Y) expression. As a consequence, daily intraperitoneal injection increased body weight, body fat content, food intake, liver size and serum insulin levels. All of these characteristics resemble the phenotype of leptin and LR-deficient animals. The results of the present study support proposed models of the activated LR complex, and demonstrate that it is possible to block LR signalling without affecting ligand binding. These nanobodies form new tools to study the mechanisms of BBB (blood-brain barrier) leptin transport and the effect of LR inhibition in disease models.
脂肪细胞衍生细胞因子瘦素充当代谢开关，将身体的代谢与高能量消耗过程（如生殖和免疫反应）联系起来。越来越多的证据表明，瘦素在人类疾病中发挥作用，如自身免疫性疾病和癌症，从而为开发瘦素拮抗剂提供了依据。在本研究中，我们生成并评估了一组针对 LR（瘦素受体）的中和纳米抗体。纳米抗体由骆驼科成员中天然存在的单链抗体的可变结构域组成。我们鉴定了针对不同 LR 亚结构域的三类中和纳米抗体：即 CRH2（细胞因子受体同源 2）、Ig 样和 FNIII（纤连蛋白 III）结构域。只有针对 CRH2 结构域的纳米抗体能抑制瘦素结合。我们可以证明，针对 Ig 样结构域的纳米抗体能有效干扰瘦素依赖性调节下丘脑 NPY（神经肽 Y）表达。结果，每天腹腔注射增加了体重、体脂肪含量、食物摄入、肝脏大小和血清胰岛素水平。所有这些特征都类似于瘦素和 LR 缺陷动物的表型。本研究的结果支持激活的 LR 复合物的提出模型，并证明在不影响配体结合的情况下，阻断 LR 信号是可能的。这些纳米抗体形成了新的工具，可用于研究 BBB（血脑屏障）瘦素转运的机制和 LR 抑制在疾病模型中的作用。
