Hepatocyte Stress Responses to Metabolic Modulators

Primary Human Hepatocytes (HH, Lonza, Walkersville, MD, USA) were cultured in Hepatocyte Culture Medium (Lonza) according to the manufacturer’s protocol. HepG2 obtained from ATCC (Sigma-Aldrich, St Louis, MO, USA) were grown in CO₂ (5%) incubator at 37 °C in high glucose Dulbecco’s modified Eagle’s medium (DMEM) GlutaMAX Supplement (Thermo Fisher Scientific, San Jose, CA, USA) after addition of 10% (v/v) fetal bovine serum, 100 U penicillin, and 100 μg/mL streptomycin. Human cell lines were tested periodically for mycoplasma by using MycoAlert PLUS detection kit (Lonza). Cells were treated with 500 µM hydroxycitrate (HCA, Sigma-Aldrich) or 200 µg/mL red wine powder (RWP) obtained as previously described from the Italian red wine Aglianico del Vulture [21 (link)]. After 1 h of treatment with HCA or RWP, cells were stimulated with 5 ng/mL of TNFα (Sigma-Aldrich) for up to 24 h. Where indicated, cells were treated also with 20 μM IKK inhibitor VII (IKK 16, Sigma-Aldrich), 5 mM sodium acetate (Ac, Sigma-Aldrich) or 5 mM sodium malate (M, Sigma-Aldrich) alone or in combination with 500 μM NADPH (Sigma-Aldrich).