Dataset Information

Development of an In Vitro Metabolic Dysfunction-Associated Steatohepatitis Model to Investigate Altered Drug Metabolizing Enzymes, Transport Proteins, and Hepatobiliary Disposition

ABSTRACT: Metabolic dysfunction-associated steatotic liver disease (MASLD) is estimated to affect approximately 30% of adults worldwide. The progressive form of MASLD, namely metabolic-dysfunction steatohepatitis (MASH), is a leading cause of chronic liver disease. MASH is marked by hepatocellular fat accumulation (steatosis), ballooning, and inflammation. Although many in vitro and in vivo models replicate MASH pathophysiology, no in vitro hepatocyte MASH model has been evaluated for its ability to reflect clinically observed changes in drug metabolizing enzyme (DME) and transporter characteristics such as abundance and activity. This study addressed this knowledge gap by developing a model using sandwich-cultured human hepatocytes (SCHH) that mimics both MASH pathophysiology and alterations in DME and transporter concentrations and function. Lipid-cytokine treatments were first optimized using differentiated HuH-7 cells based on cellular toxicity profiles and their ability to induce a MASH-like phenotype. Three final treatments, all including TNF-α (1 ng/mL) and IL-6 (1.2 ng/mL), were selected for evaluation in SCHH: (1) oleic acid (OA):palmitic acid (PA) (1:2, 0.5 mM), (2) a lipid mix (lysophospholipids mixture + OA:PA), and (3) lipid mix+0.01 mM cholesterol. Treatments were incubated for 72 h with SCHH from three donors. Quantitative targeted absolute proteomics (QTAP), employing stable isotope labeled (SIL) peptide standards and microLC-MS/MS measurement, was used to quantify transporter and DME concentrations in the SCHH total membrane fraction. Concentrations of ten transporters, ten CYP450s, seven UGTs and five other DMEs could be measured above the lower limit of quantification of 0.1 pmol/mg membrane protein. B-CLEAR® technology was used, also, to evaluate transporter function with [3H]-taurocholate (TCA) and [3H]-estradiol-17β-glucuronide (E217G) employed as probe substrates of function. All three treatments significantly increased lipid droplet formation and peroxidation, characteristics of MASH, in SCHH with minimal toxicity. The treatments also altered DME and transporter concentrations in a similar manner to changes observed in liver tissue from patients with MASH. Across treatments, concentrations of bile salt export pump (BSEP), sodium taurocholate co-transporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, and multidrug resistance-associated protein (MRP) 2 were reduced by 0.66–0.57-fold, 0.71–0.52-fold, 0.74–0.63-fold, 0.82–0.80-fold, and 0.71–0.48-fold, respectively. Correspondingly, TCA apparent uptake clearance and biliary clearance were reduced by 0.70–0.26-fold and 0.61–0.27-fold, respectively. E217G apparent uptake clearance was reduced by 0.67–0.35-fold while biliary excretion index values for E217G were reduced to negligible levels. Overall, the findings demonstrate that lipid–cytokine treatments induce MASH-like changes in SCHH, including clinically relevant reductions in DME and transporter concentrations and transporter function. This model may serve as a valuable tool for predicting altered hepatobiliary drug disposition in MASH.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hepatocyte, Liver

DISEASE(S): Non-alcoholic Steatohepatitis

SUBMITTER: John Fallon

LAB HEAD: Kim Brouwer

PROVIDER: PXD071083 | Pride | 2025-12-12

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	SCHH_Digests_21st_December_2023.timeseries.data	Other
	SCHH_Digests_21st_December_2023.wiff	Wiff
	SCHH_Digests_21st_December_2023.wiff.scan	Wiff
	SCHH_Digests_21st_December_2023.wiff2	Wiff
	SCHH__WAM__Digests_21st_December_2023__MASH_Model_.qsession	Other

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Project description:Rates of hepatocellular carcinoma (HCC) are increasing rapidly due to the epidemic of metabolic dysfunction-associated steatohepatitis (MASH). In addition to increased incidence, emerging evidence suggests that MASH driven HCC is associated with poor survival outcomes potentially due to the complex liver microenvironment which is characterized by hypoxia, steatosis, and fibrosis. Lenvatinib, is a multi-tyrosine kinase inhibitor, that is a standard of care therapy for unresected HCC, but 5-year survival rates are less than 20%. Therefore, developing treatments that inhibit cancer growth kinetics and target the tumor microenvironment to improve the therapeutic response in MASH-HCC are needed. Salsalate is a rheumatoid arthritis therapy that stimulates the AMP-activated protein kinase (AMPK) increasing fatty acid oxidation while reducing de-novo lipogenesis, fibrosis and cell proliferation pathways. Thus, we hypothesized that Salsalate could improve the therapeutic efficacy of Lenvatinib in MASH-HCC. In the current study, we show that treatment of human HCC cells with clinically relevant concentrations of Lenvatinib and Salsalate synergistically suppress proliferation and clonogenic survival, activate AMPK and inhibit the mTOR-HIF1a and Erk1/2 signaling pathways. In orthotopic xenograft and MASH-HCC mouse models Lenvatinib and Salsalate combination therapy suppressed angiogenesis and steatosis and fibrosis. RNA-sequencing revealed combination therapy enhanced mitochondria fatty acid oxidation and suppressed glycolysis, angiogenesis, fibrosis and cell cycle progression with regulatory network analysis suggesting a potential role for Activating transcription factor 3 (ATF3) and ETS-proto-oncogene-1 (ETS1). These data suggest that Lenvatinib and Salsalate combination therapy may have therapeutic potential for MASH-HCC due to effective metabolic rewiring and growth inhibition, leading to improvements in the liver microenvironment and inhibition of HCC proliferation.

Project description:Metabolic Dysfunction Associated Steatohepatitis (MASH) is characterized by excess circulating toxic lipids, hepatic steatosis, and liver inflammation. Monocyte adhesion to the liver sinusoidal endothelial cells (LSEC) and transendothelial migration (TEM) are crucial in the inflammatory process. LSEC under lipotoxic stress develops a pro-inflammatory phenotype known as endotheliopathy. However, the mediators of endotheliopathy remain obscure. Primary mouse LSEC isolated from C57BL/6J mice on chow or MASH-inducing diets rich in fat, fructose, and cholesterol (FFC) were subjected to multi-omics profiling. Mice with established MASH, due to a choline-deficient high-fat diet (CDHFD) or FFC diet, were treated with two structurally distinct GSK3 inhibitors [LY2090314, elraglusib (9-ING-41)]. Integrated pathway analysis of mouse LSEC proteome and transcriptome indicates that leukocyte TEM and focal adhesion are major pathways altered in MASH. Kinome profiling of the LSEC phospho-proteome identified GSK3β as the major kinase hub in MASH. GSK3β activating phosphorylation was increased in primary human LSEC treated with the toxic lipid palmitate and in human MASH. Palmitate upregulated the expression of C-X-C motif chemokine ligand 2, intracellular adhesion molecule 1, and phosphorylated focal adhesion kinase, via a GSK3-dependant mechanism. Congruently, the adhesive and transendothelial migratory capacities of primary human neutrophils and THP-1 monocytes through LSEC monolayer under lipotoxic stress were reduced by GSK3 inhibition. Treatment with the GSK3 inhibitors LY2090314 and elraglusib ameliorated liver inflammation, injury, and fibrosis in FFC and CDHFD-fed mice, respectively. Immunophenotyping of intrahepatic leukocytes from CDHFD-fed mice treated with elraglusib using cytometry by the time of flight showed reduced proinflammatory monocyte-derived macrophages and monocyte-derived dendritic cells infiltration.GSK3 inhibition attenuates lipotoxicity-induced LSEC endotheliopathy and may serve as a potential therapeutic strategy in human MASH.

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Development of an In Vitro Metabolic Dysfunction-Associated Steatohepatitis Model to Investigate Altered Drug Metabolizing Enzymes, Transport Proteins, and Hepatobiliary Disposition

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