Figure 2

GABA enhances IGF-1 release, leading to an increase in growth rate via IGF-1R. (A) Preosteoblast MC3T3-E1 cells (1 × 10⁴ cells/mL) were treated with 10 mM GABA for 14 days (D), and the media were replaced every three days with GABA. (B) In a parallel experiment, MC3T3-E1 cells were treated with GABA (0–10 mM) and 2 mM β-glycerophosphate (GP) for seven days. Total RNA was extracted at the indicated time points, and cDNA was synthesized. RT-PCR was performed to determine the expression level of IGF-1 and IGF-1R. Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) was used as an internal control. The expression of IGF-1 and IGF-1R relative to that of GAPDH level was determined (bottom). (C) To evaluate extracellular IGF-1 release, MC3T3-E1 cells (1 × 10⁴ cells/mL) were treated with GABA (0–10 mM) and 2 mM GP for seven days. Cell culture media were collected, and colorimetric enzyme-linked immunosorbent assay (ELISA) was performed. (D,E) Zebrafish larvae (n = 20) at three days post-fertilization (dpf) were pretreated with 10 µM picropodophyllin (PPP) 2 h before treatment with GABA (0–25 mM). (D) RT-PCR was performed to determine the expression of IGF-1. The expression of IGF-1 relative to that of zβ-actin level was determined (bottom). (E) Total body length was measured at 9 dpf using a stereomicroscope (×4). Graph represents the total body length of zebrafish larvae (right). Significant differences among the groups were determined using Student’s t-test and one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± standard error of the mean (* p < 0.05, ** p < 0.01, and *** p < 0.001).