Hepatology
Abstract
Metabolic dysfunction–associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide for which there is only one approved treatment. Adenosine monophosphate–activated protein kinase (AMPK) is an interesting therapeutic target since it acts as a central regulator of cellular metabolism. Despite efforts to target AMPK, no direct activators have yet been approved for treatment of this disease. This study investigated the effect of the AMPK activator ATX-304 in a preclinical mouse model of progressive fatty liver disease. The data demonstrated that ATX-304 diminishes body fat mass, lowers blood cholesterol levels, and mitigates general liver steatosis and the development of liver fibrosis, but with pronounced local heterogeneities. The beneficial effects of ATX-304 treatment were accompanied by a shift in the liver metabolic program, including increased fatty acid oxidation, reduced lipid synthesis, as well as remodeling of cholesterol and lipid transport. We also observed variations in lipid distribution among liver lobes in response to ATX-304, and a shift in the zonal distribution of lipid droplets upon treatment. Taken together, our data suggested that ATX-304 holds promise as a potential treatment for MASLD.
Authors
Emanuel Holm, Isabeau Vermeulen, Saba Parween, Ana López-Pérez, Berta Cillero-Pastor, Michiel Vandenbosch, Silvia Remeseiro, Andreas Hörnblad
Abstract
BACKGROUND. Alterations in circulating metabolites have been described in obese metabolic dysfunction-associated steatotic liver disease (MASLD), but data on lean MASLD are lacking. We investigated serum metabolites, including microbial bile acids (BAs) and short-chain fatty acids (SCFAs), and their association with lean and obese MASLD. METHODS. Serum samples from 204 subjects of European descent were allocated to four groups: lean healthy (n=61), lean MASLD (n=49), obese healthy (n=47) and obese MASLD (n=47). LC/MS-based metabolomics was performed followed by linear model analysis. MASLD prediction was assessed based on LASSO regression. Functional effects of significantly altered molecules were confirmed in organotypic 3D primary human liver cultures. RESULTS. Lean MASLD was characterized by elevated isobutyrate, along with higher methionine sulfoxide, propionate and phosphatidylcholines. Patients with obese MASLD had increased sarcosine and decreased lysine and asymmetric dimethylarginine. Using metabolites, sex and body mass index, MASLD vs. healthy could be predicted with a median AUC of 86.5% and 85.6% in the lean and obese subgroups, respectively. Functional experiments in organotypic 3D primary human liver cultures showed that propionate and isobutyrate induced lipid accumulation and altered expression of genes involved in lipid and glucose metabolism. CONCLUSION. Our results indicate that lean MASLD is characterized by a distinct metabolite pattern related to amino acid metabolism, lipids and SCFAs, while metabolic pathways of lipid accumulation are differentially activated by microbial metabolites. Our findings highlight an important role of microbial metabolites in MASLD pathogenesis, with implications for the predictive and mechanistic assessment of liver disease across different weight categories. FUNDING. The work received funding from the Robert Bosch Stiftung, Stuttgart, Germany, the Swedish Research Council [grant numbers 2021-02801, 2023-03015 and 2024-03401], the ERC Consolidator Grant 3DMASH [101170408], Ruth and Richard Julin Foundation for Gastroenterology [grant number 2021-00158], the SciLifeLab and Wallenberg National Program for Data-Driven Life Science [WASPDDLS22:006], and the Novo Nordisk Foundation [NNF23OC0085944 and NNF23OC0084420]. JT was supported by PMU-FFF [grant number E-18/28/148-FEL].
Authors
Mathias Haag, Stefan Winter, Aurino M. Kemas, Julia Tevini, Alexandra Feldman, Sebastian K. Eder, Thomas K. Felder, Christian Datz, Bernhard Paulweber, Gerhard Liebisch, Oliver Burk, Volker M. Lauschke, Elmar Aigner, Matthias Schwab
Abstract
Metabolic dysfunction-associated steatotic liver disease (MASLD)—characterized by excess accumulation of fat in the liver—now affects one third of the world’s population. As MASLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply characterized cohort of patients spanning the full histopathologic spectrum of MASLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of MASLD-associated liver fibrosis.
Authors
Sebastian Steinhauser, David Estoppey, Dennis P. Buehler, Yanhua Xiong, Nicolas Pizzato, Amandine Rietsch, Fabian Wu, Nelly Leroy, Tiffany Wunderlin, Isabelle Claerr, Philipp Tropberger, Miriam Müller, Alexandra Vissieres, Lindsay M. Davison, Eric H. Farber-Eger, Quinn S. Wells, Quanhu Sheng, Sebastian Bergling, Sophia A Wild, Pierre Moulin, Jiancong Liang, Wayne J. English, Brandon Williams, Judith Knehr, Marc Altorfer, Alejandro Reyes, Johannes Voshol, Craig Mickanin, Dominic Hoepfner, Florian Nigsch, Mathias Frederiksen, Charles R. Flynn, Barna D. Fodor, Jonathan D. Brown, Christian Kolter
Abstract
The functional plasticity of tumor-infiltrating B (TIL-B) cells spans from anti-tumor responses to non-canonical immune suppression. Yet, how tumor microenvironment (TME) influences TIL-B development is still underappreciated. Our current study integrated single cell transcriptomics and BCR (B cell receptor) sequencing to profile TIL-B phenotypes and clonalities in hepatocellular carcinoma (HCC). Using trajectory and gene regulatory network analysis, we were able to characterize plasma cells, memory and naïve B cells within the HCC TME and further revealed a downregulation of BCR-signaling genes in plasma cells and a subset of inflammatory TNF+ memory B cells. Within the TME, non-switch memory B cell subset acquires an age-associated B cell phenotype (TBET+, CD11c+) and expressed higher levels of PD-L1, CD25 and granzyme B. We further demonstrated that the presence of HCC tumor cells could confer suppressive functions on peripheral blood B cells which in turn, dampen T cell co-stimulation. To the best of our knowledge, these findings represent novel mechanisms of non-canonical immune suppression in HCC. While previous studies identified atypical memory B cells in chronic hepatitis and across several solid cancer types, we further highlighted their potential role as regulatory B cells (Bregs) within both the TME and peripheral blood of HCC patients.
Authors
Shi Yong Neo, Timothy Wai Ho Shuen, Shruti Khare, Joni Chong, Maichan Lau, Niranjan Shirgaonkar, Levene Chua, Junzhe Zhao, Keene Lee, Charmaine Tan, Rebecca Ba, Janice Lim, Joelle Chua, Hui Shi Cheong, Hui Min Chai, Chung Yip Chan, Alexander Yaw Fui Chung, Peng Chung Cheow, Prema Raj Jeyaraj, Jin Yao Teo, Ye Xin Koh, Aik Yong Chok, Pierce Kah Hoe Chow, Brian Goh, Wei Keat Wan, Wei Qiang Leow, Tracy Jie Zhen Loh, Po Yin Tang, Jayanthi Karunanithi, Nye Thane Ngo, Tony Kiat Hon Lim, Shengli Xu, Ramanuj Dasgupta, Han Chong Toh, Kong-Peng Lam
Abstract
Authors
Jing Zeng, Derrick Zhao, Grayson Way, Andrew Fagan, Michael Fuchs, Puneet Puri, Brian C. Davis, Xuan Wang, Emily C. Gurley, Phillip B. Hylemon, Jian-Gao Fan, Masoumeh Sikaroodi, Patrick M. Gillevet, Huiping Zhou, Jasmohan S. Bajaj
Abstract
Due to the limitations of available in vitro systems and animal models, we lack a detailed understanding of the pathogenetic mechanisms and have minimal treatment options for liver fibrosis. Therefore, we engineered a live cell imaging system that assesses fibrosis in a human multi-lineage hepatic organoid in a microwell (i.e., microHOs). Transcriptomic analysis revealed that TGFβ1 converted mesenchymal cells in microHOs into myofibroblast-like cells resembling those in fibrotic human liver tissue. When pro-fibrotic intracellular signaling pathways were examined, the anti-fibrotic effect of receptor-specific tyrosine kinase inhibitors was limited to the fibrosis induced by the corresponding growth factor, which indicates their anti-fibrotic efficacy would be limited to fibrotic diseases solely mediated by that growth factor. Based upon transcriptomic and transcription factor activation analyses in microHOs, GSK3β and p38 MAPK inhibitors were identified as potential new broad-spectrum therapies for liver fibrosis. Other new therapies could subsequently be identified using the microHO system.
Authors
Yuan Guan, Zhuoqing Fang, Angelina Hu, Sarah Roberts, Meiyue Wang, Wenlong Ren, Patrik K. Johansson, Sarah C. Heilshorn, Annika Enejder, Gary Peltz
Abstract
Biliary obstruction and cholangiocyte hyperproliferation are important features of cholangiopathies affecting the large extrahepatic bile duct (EHBD). The mechanisms underlying obstruction-induced cholangiocyte proliferation in the EHBD remain poorly understood. Developmental pathways, including WNT signaling, are implicated in regulating injury responses in many tissues, including the liver. To investigate the contribution of WNT signaling to obstruction-induced cholangiocyte proliferation in the EHBD, we used complementary in vivo and in vitro models with pharmacologic interventions and transcriptomic analyses. To model obstruction, we used bile duct ligation (BDL) in mice. Human and mouse biliary organoids and mouse biliary explants were used to investigate the effects of WNT activation and inhibition in vitro. We observed an upregulation of WNT ligand expression associated with increased biliary proliferation following obstruction. Cholangiocytes were identified as both WNT ligand-expressing and WNT responsive cells. Inhibition of WNT signaling decreased cholangiocyte proliferation in vivo and in vitro, while activation increased proliferation. WNT effects on cholangiocyte proliferation were β-catenin-dependent, and we showed a direct effect of WNT7B on cholangiocyte growth. Our studies suggested that cholangiocyte-derived WNT ligands can activate WNT signaling to induce proliferation after obstructive injury. These findings implicated the WNT pathway in injury-induced cholangiocyte proliferation within the EHBD.
Authors
Ashley N. Calder, Mirabelle Q. Peter, John W. Tobias, Nureen H. Mohamad Zaki, Theresa M. Keeley, Timothy L. Frankel, Linda C. Samuelson, Nataliya Razumilava
Abstract
Hepatic macrophages and regulatory T cells (Tregs) play an important role in the maintenance of liver immune homeostasis, but the mechanism by which hepatic macrophages regulate Tregs in acute liver injury remains largely unknown. Here, we found that the hepatic Treg proportion and β-catenin expression in hepatic macrophages were associated with acetaminophen (APAP) and D-galactosamine (D-GalN)/ lipopolysaccharide (LPS)-induced acute liver injury. Interestingly, β-catenin was markedly upregulated only in infiltrating macrophages, but not in resident Kupffer cells. Myeloid-specific β-catenin knockout mice showed an increased inflammatory cell infiltration and hepatocyte apoptosis. Moreover, myeloid β-catenin deficiency decreased the hepatic Treg proportion in the injured liver. Mechanistically, in vitro co-culture experiments revealed that macrophage β-catenin modulated its exosome composition, and influenced Treg differentiation. Using mass spectrometry-based proteomics, we identified that macrophage β-catenin activation increased the level of exosomal α-SNAP, which in turn promoted Treg differentiation. Overall, our findings demonstrated a molecular mechanism that macrophage β-catenin regulated the Treg proportion in the liver by enhancing the expression of exosomal α-SNAP, providing insights into the pathophysiology of acute liver injury.
Authors
Ruobin Zong, Yujie Liu, Mengya Zhang, Buwei Liu, Wei Zhang, Hankun Hu, Changyong Li
Abstract
Excessive fructose intake is a risk factor for the development of obesity and its complications. Targeting ketohexokinase (KHK), the first enzyme of fructose metabolism, has been investigated for the management of MASLD. We compared the effects of systemic, small molecule inhibitor of KHK enzymatic activity to hepatocyte-specific, GalNAc-siRNA mediated knockdown of KHK in mice on a HFD. We measured KHK enzymatic activity, extensively quantified glycogen accumulation, performed RNAseq analysis, and enumerated hepatic metabolites using mass spectrometry. Both KHK siRNA and KHK inhibitor led to an improvement in liver steatosis, however, via substantially different mechanisms. KHK knockdown decreased the de novo lipogenesis pathway, whereas the inhibitor increased the fatty acid oxidation pathway. Moreover, KHK knockdown completely prevented hepatic fructolysis and improved glucose tolerance. Conversely, the KHK inhibitor only partially reduced fructolysis, but it also targeted triokinase, mediating the third step of fructolysis. This leads to the accumulation of fructose-1 phosphate, resulting in glycogen accumulation, hepatomegaly, and impaired glucose tolerance. Overexpression of wild-type, but not kinase-dead KHK in cultured hepatocytes increased hepatocyte injury and glycogen accumulation when treated with fructose. The differences between KHK inhibition and knockdown are, in part, explained by the kinase-dependent and independent effects of KHK on hepatic metabolism.
Authors
Se-Hyung Park, Taghreed Fadhul, Lindsey R. Conroy, Harrison A. Clarke, Ramon C. Sun, Kristina Wallenius, Jeremie Boucher, Gavin O'Mahony, Alessandro Boianelli, Marie Persson, Sunhee Jung, Cholsoon Jang, Analia S. Loria, Genesee J. Martinez, Zachary A. Kipp, Evelyn A. Bates, Terry D. Hinds, Jr., Senad Divanovic, Samir Softic
Abstract
Cell cycle inhibitors have a long history as cancer treatment. Here, we reported that these inhibitors combated cancer partially via Stimulator of IFN genes (STING) signaling pathway. We demonstrated that Paclitaxel (microtubule stabilizer), Palbociclib (cyclin dependent kinase 4/6 inhibitor), AZD1152 and GSK1070916 (aurora kinase B inhibitors) have anti-cancer functions beyond arresting cell cycle. They consistently caused cytosolic DNA accumulation and DNA damage, which inadvertently triggered the cytosolic DNA sensor DEAD-box helicase 41 (DDX41) and activated STING to secrete pro-inflammatory senescence-associated secretory phenotype factors (SASPs). Interestingly, we found that DDX41 was a transcriptional target of HIF. Hypoxia induced expression of DDX41 through HIF-1, making hypoxic HCC cells more sensitive to the anti-mitotic agents in STING activation and SASP production. The SASPs triggered immune cell infiltration in tumors for cancer clearance. The treatment of cell cycle inhibitors, especially Paclitaxel, extends survival by perturbing mouse HCC growth when used in combination with anti-PD-1. We observed a trend that Paclitaxel suppressed STINGWT HCC more effectively than STINGKO HCC, suggesting that STING might contribute to the anti-tumor effects of Paclitaxel. Our study revealed the immune-mediated tumor-suppressing properties of cell cycle inhibitors and suggested combined treatment with immunotherapy as a potential therapeutic approach.
Authors
Po Yee Wong, Cerise Yuen Ki Chan, Helen Do Gai Xue, Chi Ching Goh, Jacinth Wing Sum Cheu, Aki Pui Wah Tse, Misty Shuo Zhang, Yan Zhang, Carmen Chak Lui Wong
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