Honokiol acts as an AMPK complex agonist therapeutic in non-alcoholic fatty liver disease and metabolic syndrome

**Background:** Non-alcoholic fatty liver disease (NAFLD) and its related metabolic syndrome are major health threats with no approved pharmacological therapies. AMP-activated protein kinase (AMPK) is a key energy sensor, and activating AMPK has shown promise against NAFLD, but chronic pan-AMPK activation may cause adverse effects. The AMPKγ1 subunit is a potentially safer therapeutic target. Honokiol, a neolignan biphenol from magnolia plants, has anti-inflammatory and anti-oxidative properties but its role in NAFLD and mechanism of action were not fully understood.

**Methods:** The authors screened an FDA-approved drug library of approximately 3000 compounds using L02 human hepatocytes exposed to palmitic acid/oleic acid (PO) to induce lipid accumulation. Honokiol was identified as a top candidate. Its therapeutic effects were evaluated in primary mouse hepatocytes and in three mouse models: high-fat diet (HFD)-induced NAFLD (12 weeks HFD then 12 weeks treatment), choline-deficient L-amino acid-defined high-fat diet (CDAHFD)-induced NASH (1 week diet then 3 weeks treatment), and methionine-choline deficient (MCD) diet-induced NASH (same schedule). Honokiol was administered at 100 mg/kg/day by oral gavage. Outcomes included body/liver/adipose weights, histopathology (HE, oil red O, picrosirius red, CD11b IHC), serum ALT/AST/TC/TG, liver TG/TC/NEFA, glucose tolerance tests (GTT), and insulin tolerance tests (ITT). Transcriptomic analysis (RNA-seq) was performed on primary hepatocytes and liver/WAT tissues. Mechanistic studies used compound C (AMPK inhibitor, 10 mg/kg i.p. every other day), PRKAA1/2 double-knockout (DKO) hepatocytes, PRKAG1 (AMPKγ1) knockdown, and biotin-streptavidin binding assays with wild-type and mutant AMPKγ1.

**Key Results:** Honokiol significantly reduced lipid accumulation in primary hepatocytes (BODIPY staining, TG, TC). In HFD-fed mice, honokiol decreased body weight, liver weight, hepatic lipid droplets, fibrosis, and CD11b+ cell infiltration. Liver TG, TC, and NEFA were reduced, as were serum ALT, AST, TC, and TG. No adverse effects on heart, kidney, or spleen were observed. Honokiol improved glucose tolerance (GTT) and insulin tolerance (ITT) and reduced serum insulin. In CDAHFD and MCD NASH models, honokiol similarly reduced body/liver weight, hepatic steatosis, fibrosis, inflammation, and serum ALT/AST/TG. Transcriptomics showed upregulation of AMPK signaling and fatty acid degradation pathways, and downregulation of inflammation and fibrosis pathways. Western blotting confirmed AMPKα and ACC phosphorylation and mTOR inhibition in vitro and in vivo. Compound C co-treatment and PRKAA1/2 DKO abolished honokiol's lipid-lowering effects. Honokiol did not affect classical AMPK upstream regulators (LKB1, CAMKK2, TAK1, PP2C) or cellular ATP/ADP/AMP levels. Biotin-avidin binding assays demonstrated direct binding of honokiol to AMPKγ1 but not AMPKγ2. Mutation of three binding sites (H151, R152, K243) on AMPKγ1 (AMPKγ1-3A) largely abolished this interaction. PRKAG1 knockdown reduced honokiol-induced AMPK activation and lipid lowering, which was rescued by wild-type AMPKγ1 but not the 3A mutant.

**Clinical Implications:** This study identifies honokiol as a promising drug candidate for the full spectrum of NAFLD and metabolic syndrome, acting through a novel mechanism—direct binding to AMPKγ1 to activate AMPK signaling. This mechanism avoids classical upstream regulators and may circumvent side effects associated with pan-AMPK activation (e.g., cardiac hypertrophy). The use of an FDA-approved drug library screening approach leverages known safety profiles, potentially accelerating clinical translation. However, these findings are preclinical; human studies are needed to confirm efficacy and safety in patients with NAFLD/NASH.