Nuclear receptor ligand screening in an iPSC-derived in vitro blood–brain barrier model identifies new contributors to leptin transport

Abstract

Background The hormone leptin exerts its function in the brain to reduce food intake and increase energy expenditure to prevent obesity. However, most obese subjects reflect the resistance to leptin even with elevated serum leptin. Considering that leptin must cross the blood–brain barrier (BBB) in several regions to enter the brain parenchyma, altered leptin transport through the BBB might play an important role in leptin resistance and other biological conditions. Here, we report the use of a human induced pluripotent stem cell (iPSC)-derived BBB model to explore mechanisms that influence leptin transport. Methods iPSCs were differentiated into brain microvascular endothelial cell (BMEC)-like cells using standard methods. BMEC-like cells were cultured in Transwell filters, treated with ligands from a nuclear receptor agonist library, and assayed for leptin transport using an enzyme-linked immune sorbent assay. RNA sequencing was further used to identify differentially regulated genes and pathways. The role of a select hit in leptin transport was tested with the competitive substrate assay and after gene knockdown using CRISPR techniques. Results Following a screen of 73 compounds, 17β-estradiol was identified as a compound that could significantly increase leptin transport. RNA sequencing revealed many differentially expressed transmembrane transporters after 17β-estradiol treatment. Of these, cationic amino acid transporter-1 (CAT-1, encoded by SLC7A1) was selected for follow-up analyses due to its high and selective expression in BMECs in vivo. Treatment of BMEC-like cells with CAT-1 substrates, as well as knockdown of CAT-1 expression via CRISPR-mediated epigenome editing, yielded significant increases in leptin transport. Conclusions A major female sex hormone, as well as an amino acid transporter, were revealed as regulators of leptin BBB transport in the iPSC-derived BBB model. Outcomes from this work provide insights into regulation of hormone transport across the BBB.

Syndromic and Monogenic Obesity: New Opportunities Due to Genetic-Based Pharmacological Treatment

“…With the iPSC methodology, brown adipose progenitor cells can be used for both cell transplantation as well as for anti-obesity drug discovery. In vitro representations of human genetic conditions can also improve the examination of the molecular and cellular impact of certain genetic mutations in energy homeostasis pathways [97][98][99][100][101][102].…”

Recent Advances in the Knowledge of the Mechanisms of Leptin Physiology and Actions in Neurological and Metabolic Pathologies

“…The recent characterization of new factors such as the cationic amino acid transporter-1, that increases leptin transport, suggests additional mechanisms for leptin transport. In this regard, the treatment of brain microvascular endothelial cell-like cells with substrates for this transporter generated a significant increase in leptin transport [ 105 ]. All of these factors mentioned above are involved in changes in the expression of the different neuropeptides that regulate energy intake and homeostasis, and in a prolonged situation, this causes metabolic disorders by disrupting the crosstalk between the hypothalamus and periphery, a situation that can be reversed with some antilipemic drugs that reduce body weight [ 106 ].…”

Evidence for an alternative insulin transporter at the blood-brain barrier

“…Recently, it was identified in a high-throughput screen that the amino acid transporter, SLC7A1, also known as CAT-1, could regulate leptin transport across an iPSC-derived BEC model [92]. This raises as a possibility that the same, or another amino acid transporter, could regulate insulin transport across the BBB.…”