Positive allosteric γ‐aminobutyric acid type A receptor modulation prevents lipotoxicity‐induced injury in hepatocytes in vitro

Abstract

Aim: To determine if a novel positive allosteric modulator of the γ-aminobutyric acid type A (GABA A ) receptor, the thioacrylamide-derivative HK4, which does not penetrate the blood-brain barrier, protects human hepatocytes against lipotoxicityinduced injury.Materials and Methods: Allosteric modulation of the GABA A receptor by HK4 was determined by patch clamp in HEK-293 cells, calcium influx in INS-1E cells and by using the specific GABA A channel blockers picrotoxin and tert-butylbicyclophosphorothionate (TBPS) in HepG2 cells. Apoptosis was analysed using caspase 3/7, terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) and array assays in HepG2 cells and/or human primary hepatocytes. Phosphorylation of STAT3 and the NF-κB subunit p65, protein disulphide isomerase (PDI) and poly-ADP-ribose polymerase-1 (PARP-1) was detected by Western blotting.Results: Patch clamping, calcium influx measurements and apoptosis assays with the non-competitive GABA A channel blockers picrotoxin and TBPS proved HK4 as a selective positive allosteric modulator of the GABA A receptor. In HepG2 cells, which expressed the main GABA A receptor subunits, HK4 prevented palmitate-induced apoptosis. This protective effect was mediated by downregulation of caspase 3/7 activity and was additionally verified by TUNEL assay. HK4 effectively prevented palmitate-induced apoptosis in human primary hepatocytes. HK4 reduced STAT3 and

Positive allosteric GABAA receptor modulation counteracts lipotoxicity-induced gene expression changes in hepatocytes in vitro

“…Substantial evidence supports that activation of the transcription factor NF-κB and downstream inflammatory signalling pathways are involved in hepatic insulin resistance (Tilg et al, 2017). In line, upregulated NF-κB gene expression in PA-treated HepG2 cells compared to untreated (p = 0.004) or PA and HK4-cotreated cells (p = 0.089) fits with protein expression analysis of our previous study (Rohbeck et al, 2022). Also in line with our previous study, the current data indicate an involvement of STAT signaling, since STAT5A is differentially expressed in our dataset.…”

“…Therefore, we aimed to determine if the positive allosteric modulator (PAM) of the GABA A receptor, HK4, could prevent the deleterious effects of lipotoxicity in hepatocytes. In our recent publication ( Rohbeck et al, 2022 ) we were able to confirm the presence of the GABA A receptor in HepG2, as described by Minuk and colleagues ( Minuk et al, 1987 ; Rohbeck et al, 2022 ). Moreover, we showed that the GABA A receptor PAM, HK4, could reduce palmitate (PA)-induced DNA fragmentation, inflammation, cell death and especially apoptosis, in vitro ( Rohbeck et al, 2022 ).…”

“…We have taken a closer look at oxidative phosphorylation and mitochondrial dysfunction (clustered together as mitochondrial respiration), as well as protein ubiquitination, since they are the most relevant pathways according to their p -values and number of included genes. Additionally, we have focused on apoptosis, because we previously demonstrated the protective effect of HK4 on PA-induced apoptosis ( Rohbeck et al, 2022 ). Since cell cycle plays a pivotal role in our lipotoxicity model with the highest number of related DEGs ( Table 1 ) and it is closely connected to apoptosis ( Caldez et al, 2020 ), we also took cell cycle into account.…”

“…In our recent publication (Rohbeck et al, 2022) we were able to confirm the presence of the GABA A receptor in HepG2, as described by Minuk and colleagues (Minuk et al, 1987;Rohbeck et al, 2022). Moreover, we showed that the GABA A receptor PAM, HK4, could reduce palmitate (PA)-induced DNA fragmentation, inflammation, cell death and especially apoptosis, in vitro (Rohbeck et al, 2022). These effects were mediated by activation of the two transcription factors nuclear factor kappa-light-chain-enhancer of activated B Cells (NF-κB) and signal transducers and activators of transcription 3 (STAT3) (Rohbeck et al, 2022).…”

“…Moreover, we showed that the GABA A receptor PAM, HK4, could reduce palmitate (PA)-induced DNA fragmentation, inflammation, cell death and especially apoptosis, in vitro ( Rohbeck et al, 2022 ). These effects were mediated by activation of the two transcription factors nuclear factor kappa-light-chain-enhancer of activated B Cells (NF-κB) and signal transducers and activators of transcription 3 (STAT3) ( Rohbeck et al, 2022 ). Those transcription factors control the expression of a large number of downstream genes related to cell proliferation, survival, stress responses and inflammation ( He and Karin, 2011 ).…”