Objective

Our laboratory recently identified the centrally circulating α-klotho protein as a novel hypothalamic regulator of food intake and glucose metabolism in mice. The current study aimed to investigate novel molecular effectors of central α-klotho in the arcuate nucleus of the hypothalamus (ARC), while further deciphering its role regulating energy balance in both humans and mice.

Methods

Cerebrospinal fluid (CSF) was collected from 22 adults undergoing lower limb orthopedic surgeries, and correlations between body weight and α-klotho were determined using an α-klotho enzyme-linked immunosorbent assay (ELISA) kit. To investigate the effects of α-klotho on energy expenditure (EE), 2-day intracerebroventricular (ICV) treatment was performed in diet-induced obesity (DIO) mice housed in TSE Phenomaster indirect calorimetry metabolic cages. Immunohistochemical staining for cFOS and patch clamp electrophysiology were used to determine the effects of central α-klotho on proopiomelanocortin (POMC) and tyrosine hydroxylase (TH) neurons. Additional stainings were performed to determine novel roles for central α-klotho to regulate non-neuronal cell populations in the ARC. Lastly, ICV pretreatment with fibroblast growth factor receptor (FGFR) or PI3kinase inhibitors was performed to determine the intracellular signaling involved in α-klotho-mediated regulation of ARC nuclei.

Results

Obese/overweight human subjects had significantly lower CSF α-klotho concentrations compared to lean counterparts (1,044 ± 251 vs. 1616 ± 218 pmol/L, respectively). Additionally, 2 days of ICV α-klotho treatment increased EE in DIO mice. α-Klotho had no effects on TH neuron activity but elicited varied responses in POMC neurons, with 44% experiencing excitatory and 56% experiencing inhibitory effects. Inhibitor experiments identified an α-klotho→FGFR→PI3kinase signaling mechanism in the regulation of ARC POMC and NPY/AgRP neurons. Acute ICV α-klotho treatment also increased phosphorylated ERK in ARC astrocytes via FGFR signaling.

Conclusion

Our human CSF data provide the first evidence that impaired central α-klotho function may be involved in the pathophysiology of obesity. Furthermore, results in mouse models identify ARC POMC neurons and astrocytes as novel molecular effectors of central α-klotho. Overall, the current study highlights prominent roles of α-klotho→FGFR→PI3kinase signaling in the homeostatic regulation of ARC neurons and whole-body energy balance.

中文翻译：

---

中枢循环 α-klotho 与人类肥胖呈负相关并调节小鼠的弓状细胞群

客观的

我们的实验室最近将中枢循环的 α-klotho 蛋白鉴定为小鼠食物摄入和葡萄糖代谢的新型下丘脑调节剂。目前的研究旨在研究下丘脑弓状核 (ARC) 中央 α-klotho 的新型分子效应物，同时进一步解读其在人类和小鼠中调节能量平衡的作用。

方法

从 22 名接受下肢骨科手术的成年人身上收集脑脊液 (CSF)，并使用 α-klotho 酶联免疫吸附测定 (ELISA) 试剂盒确定体重与 α-klotho 之间的相关性。为了研究 α-klotho 对能量消耗 (EE) 的影响，对饲养在 TSE Phenomaster 间接量热代谢笼中的饮食诱导肥胖 (DIO) 小鼠进行了 2 天的脑室内 (ICV) 治疗。cFOS 的免疫组织化学染色和膜片钳电生理学用于确定中枢 α-klotho 对阿片黑皮素原 (POMC) 和酪氨酸羟化酶 (TH) 神经元的影响。进行了额外的染色以确定中央 α-klotho 在调节 ARC 中的非神经元细胞群方面的新作用。最后，

结果

与瘦人相比，肥胖/超重人类受试者的脑脊液 α-klotho 浓度显着降低（分别为 1,044 ± 251 与 1616 ± 218 pmol/L）。此外，2 天的 ICV α-klotho 治疗增加了 DIO 小鼠的 EE。α-Klotho 对 TH 神经元活动没有影响，但在 POMC 神经元中引起不同的反应，44% 的人经历兴奋，56% 的人经历抑制作用。抑制剂实验确定了α-klotho→FGFR→PI3激酶信号传导机制在ARC POMC和NPY/AgRP神经元的调节中。急性 ICV α-klotho 治疗还通过 FGFR 信号增加了 ARC 星形胶质细胞中的磷酸化 ERK。

结论

我们的人类 CSF 数据提供了第一个证据，表明中枢 α-klotho 功能受损可能与肥胖的病理生理学有关。此外，小鼠模型的结果将 ARC POMC 神经元和星形胶质细胞鉴定为中枢 α-klotho 的新型分子效应器。总体而言，目前的研究强调了 α-klotho→FGFR→PI3 激酶信号在 ARC 神经元的稳态调节和全身能量平衡中的突出作用。