Chronic fatty acid treatment decreased HMGCL content and ketogenesis in cultured hepatocytes. (a) β-OHB content in the culture medium at 2, 6, and 24 h post-PA and -OA treatments. (b) CD36, CPT1b, HMGCS2, HMGCL, and BDH1 contents at 2 and 24 h post-PA treatment. (c) CD36, CPT1b, HMGCS2, HMGCL, and BDH1 contents at 2 and 24 h post-OA treatment. (d) Mitochondrion-targeted antioxidants, MitoTEMPO and SS31, decreased mitochondrial ROS in untreated, PA-treated, and OA-treated hepatocytes. (e) Scavenging mitochondrial ROS using MitoTEMPO and SS31 increased β-OHB content in the culture medium at 24 h post-PA and -OA treatments. (f) Scavenging mitochondrial ROS increased the HMGCL content in cultured hepatocytes at 24 h post-PA and -OA treatments. (g) HMGCL overexpression showed no significant effects on the CD36 and CPT1b contents in hepatocytes at 24 h post-PA and -OA treatments. (h) HMGCL overexpression increased β-OHB content in the culture medium at 24 h post-PA and -OA treatments. n = 6. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.0001.