Fig. 8: Pharmacological induction of PKA-mediated phosphorylation of 17β-HSD13 at Ser33 protects against NASH.

a The chemical structure of reproterol. b Primary hepatocytes were cultured and treated with 10 μm reproterol (Rep) with or without the PKA inhibitor H89. Phosphorylated ATGL (S406), phosphorylated HSL (S660), and total HSL were immunoblotted. Forskolin (Fsk) was used as a positive control for PKA activation. c HEK293 cells were transfected with a full-length myc-tagged 17β-HSD13 WT, S33A mutant, and S33E mutant followed by IP with an anti-myc antibody. 10 μM reproterol or DMSO was added 30 min before cell lysis. Proteins retained on sepharose were blotted with an anti-phospho-(Ser/Thr) PKA substrate antibody. d Reproterol treatment increases the phosphorylation of 17β-HSD13 at serine 33 residue via PKA in vivo (see “Methods” for detail). Liver lysates were used for IP assay with an anti-myc antibody. Proteins retained on sepharose were blotted with an anti-phospho-(Ser/Thr) PKA substrate antibody. Red arrowheads indicate the phosphorylated 17β-HSD13 as a PKA-substrate. e–p Mice were fed with an HFD at 6 weeks of age for 16 weeks. Ten weeks after HFD treatment, mice began to receive reproterol treatment via intragastric administration at the dosage of 5 mg/kg body weight daily for 6 weeks. Body weight were recorded every week (e). HF-R, mice receiving both HF diet and reproterol; HF-Ctrl, mice only receiving HFD. HF-Ctrl: n = 5; HF-R: n = 5 biologically independent animals. Representative Oil red O, H&E, and Masson staining of the livers of HF-Ctrl and HF-R (f) and the NAS score (g) and fibrotic area (%) (h) in HF-Ctrl and HF-R group were shown. (scale bar, 50 μm) Liver TG (i) and TC content (j), body weight (k), and liver weight (l) in HF-Ctrl and HF-R group were measured. Expression of genes related with lipid synthesis (m), lipid transport (n), and lipid oxidation (o) was assessed. Protein expression levels of SREBP-1, SREPB-2, 17β-HSD13, ATGL, and β-actin in HF-Ctrl and HF-R groups were analyzed using immunoblot assay and quantitated (p). q Proposed mechanism by which 17β-HSD13 regulates LD lipolysis. Free fatty acids (FFAs) from extracellular and intracellular sources are packaged into triglycerides and stored in LDs. The S33-dephosphorylated 17β-HSD13 binds tightly to ATGL on the surface of LDs and sequesters CGI-58 to reduce ATGL lipolytic activity, leading to hepatocyte lipid accumulation. In contrast, upon PKA activation, the Ser33 residue of 17β-HSD13 is phosphorylated, which allows more physical interaction between ATGL and CGI-58 to increase ATGL activity, thereby reducing hepatocyte lipotoxicity. Data represent mean ± SEM; Significance was calculated by two-tailed student’s t test (g–l) or two-way ANOVA with Bonferroni post hoc analysis was performed for e, (m–p).