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

Diabetes Obes Metab. 2022 Aug;24(8):1498-1508. doi: 10.1111/dom.14719. Epub 2022 Jun 6.

Abstract

Aim: To determine if a novel positive allosteric modulator of the γ-aminobutyric acid type A (GABAA ) receptor, the thioacrylamide-derivative HK4, which does not penetrate the blood-brain barrier, protects human hepatocytes against lipotoxicity-induced injury.

Materials and methods: Allosteric modulation of the GABAA receptor by HK4 was determined by patch clamp in HEK-293 cells, calcium influx in INS-1E cells and by using the specific GABAA 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 GABAA channel blockers picrotoxin and TBPS proved HK4 as a selective positive allosteric modulator of the GABAA receptor. In HepG2 cells, which expressed the main GABAA 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 NF-κB phosphorylation, reduced cleaved PARP-1 expression and upregulated the endoplasmic reticulum (ER) chaperone PDI.

Conclusions: HK4 reduced lipotoxic-induced apoptosis by preventing inflammation, DNA damage and ER stress. We propose that the effect of HK4 is mediated by STAT3 and NF-κB. It is suggested that thioacrylamide compounds represent an innovative pharmacological tool to treat or prevent non-alcoholic steatohepatitis as first-in-class drugs.

Keywords: GABA; HK4; NAFLD; apoptosis; hepatocytes; lipotoxicity; palmitate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis
  • Calcium / metabolism
  • Caspase 3 / metabolism
  • HEK293 Cells
  • Hepatocytes
  • Humans
  • NF-kappa B / metabolism
  • NF-kappa B / pharmacology
  • Palmitates / metabolism
  • Palmitates / pharmacology
  • Picrotoxin / metabolism
  • Picrotoxin / pharmacology
  • Poly(ADP-ribose) Polymerase Inhibitors / metabolism
  • Poly(ADP-ribose) Polymerase Inhibitors / pharmacology
  • Receptors, GABA* / metabolism
  • Receptors, GABA-A* / genetics
  • Receptors, GABA-A* / metabolism
  • gamma-Aminobutyric Acid / metabolism
  • gamma-Aminobutyric Acid / pharmacology

Substances

  • NF-kappa B
  • Palmitates
  • Poly(ADP-ribose) Polymerase Inhibitors
  • Receptors, GABA
  • Receptors, GABA-A
  • Picrotoxin
  • gamma-Aminobutyric Acid
  • Caspase 3
  • Calcium