Locked Out: Phoenixin-14 Does Not Cross a Stem-Cell-Derived Blood-Brain Barrier Model

Abstract

Phoenixin-14 is a recently discovered peptide regulating appetite. Interestingly, it is expressed in the gastrointestinal tract; however, its supposed receptor, GPR173, is predominantly found in hypothalamic areas. To date, it is unknown how peripherally secreted phoenixin-14 is able to reach its centrally located receptor. To investigate whether phoenixin is able to pass the blood-brain barrier, we used an in vitro mono-culture blood-brain barrier (BBB) model consisting of brain capillary-like endothelial cells derived from human induced-pluripotent stem cells (hiPSC-BCECs). The passage of 1 nMol and 10 nMol of phoenixin-14 via the mono-culture was measured after 30, 60, 90, 120, 150, 180, 210, and 240 min using a commercial ELISA kit. The permeability coefficients (PC) of 1 nMol and 10 nMol phoenixin-14 were 0.021 ± 0.003 and 0.044 ± 0.013 µm/min, respectively. In comparison with the PC of solutes known to cross the BBB in vivo, those of phoenixin-14 in both concentrations are very low. Here, we show that phoenixin-14 alone is not able to cross the BBB, suggesting that the effects of peripherally secreted phoenixin-14 depend on a co-transport mechanism at the BBB in vivo. The mechanisms responsible for phoenixin-14's orexigenic property along the gut-brain axis warrant further research.

Keywords: appetite regulation; blood–brain barrier; brain–gut axis; gastrointestinal tract; hypothalamus; in vitro techniques; induced pluripotent stem cells; peptides; phoenixin-14.

Figure 1

Using a Millicell ERS-2 voltohmmeter equipped with a STX3 electrode transendothelial electrical resistance (TEER) in the empty hiPSC-BCEC model prior to testing with 1, 10, and 100 nMol of phoenixin-14 were assessed. No significant differences between the three concentrations were found. All data are expressed as mean ± SEM.

Figure 2

Cell viability of hiPSC-BCECs treated with phoenixin-14 was determined in comparison with an untreated control. Using CellTiter-Glo^® 2.0 Cell Viability Assay, viability of hiPSC-BCECs after incubation of 100, 10, and 1 nMol of phoenixin and 1% SDS was assessed. All data are expressed as mean ± SEM. * p < 0.05 vs. 100 nMol phoenixin-14.

Figure 3

Using an hiPSC-derived blood–brain barrier model, the permeability coefficient (PC) of 1 and 10 nMol of phoenixin-15 was assessed. No significant difference between 1 and 10 nMol of phoenixin-14 was observed. In comparison, PCs of FICT-dextran 40 kDa (known to not cross the BBB), loratadine, and caffeine (both known to cross the BBB) based on previously published data are also shown [32]. All data are expressed as mean ± SEM.