Targeting AMPK to attenuate lipid accumulation in Huh7 human hepatocytes exposed to physiological and pathophysiological concentrations of lipids

Ryan Brett; Giuseppe Deganutti; Richard Mackenzie; Leanne Hodson; Derek Renshaw; Mark C. Turner

doi:10.1530/endoabs.109.P313

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Endocrine Abstracts (2025) 109 P313 | DOI: 10.1530/endoabs.109.P313

SFEBES2025 Poster Presentations Late Breaking (68 abstracts)

Targeting AMPK to attenuate lipid accumulation in Huh7 human hepatocytes exposed to physiological and pathophysiological concentrations of lipids

Ryan Brett ¹ , Giuseppe Deganutti ¹ , Richard Mackenzie ^1,2 , Leanne Hodson ³ , Derek Renshaw ^1,2 & Mark C. Turner ^1,2

Author affiliations

¹Coventry University, Coventry, United Kingdom. ²Institute for Cardio-Metabolic Medicine, University Hospitals Coventry and Warwickshire, Coventry, United Kingdom. ³Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, United Kingdom

Introduction: Metabolic dysfunction-associated steatohepatitis (MASH) affects approximately two billion people worldwide, which if untreated, can progress to hepatocellular carcinoma and increase the risk of early mortality. Targeting 5’ AMP-activated protein kinase (AMPK) pharmacologically could attenuate lipid accumulation in hepatocytes and reduce the risk of liver disease progression. Therefore, computational modelling and in vitro experiments in the human hepatocytes model of MASH sought to establish the effects of the AMPK agonist, BI-9774, on triglyceride accumulation and cell respiration.

Methods: Computational modelling was used to establish BI-9774 binding mode to AMPK and its positive allosteric mechanism to increase AMPK activity. In vitro, experiments using Huh7 human hepatocytes were exposed to BI-9774 (1µM) for two days with increasing concentrations (0-800µM) of lipids. Immunofluorescence imaging with BODIPY was employed to detect the presence of neutral lipids. In addition, cell respiration analysis assessed the impact of BI-9774 on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR).

Results: Computational modelling showed that the phosphorylation of AMPK Ser¹⁰⁸ and BI-9774 binding have similar and cumulable effects on the dynamics of the β-subunit, suggesting an allosteric mechanism triggered by its proximity to the α-subunit. In vitro experiments showed that the total intensity of BODIPY was attenuated following BI-9774 treatment compared to control hepatocytes cultured with lipids (P< 0.01). There was an effect of BI-9774 on maximal OCR (P<0.01). In hepatocytes exposed to 800µM of lipids, BI-9774 resulted in lower basal and maximal ECAR (P<0.01).

Conclusions: These experiments show how BI-9774 binds and affects AMPK regulation and in an in vitro model exposed to pathophysiological concentrations of lipids, can attenuate triglyceride accumulation, and increase maximal OCR while lowering ECAR. Further analysis is required to determine the transcriptional and protein responses that underpin the effects of BI-9774 on HuH7 hepatocytes exposed to pathophysiological concentrations of lipids.