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Karbowska J, Kochan Z. Crosstalk Between Dietary Fatty Acids and MicroRNAs in the Regulation of Hepatic ApoB-Containing Lipoprotein Synthesis in Humans. Int J Mol Sci 2025; 26:4817. [PMID: 40429957 PMCID: PMC12112749 DOI: 10.3390/ijms26104817] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2025] [Revised: 05/10/2025] [Accepted: 05/13/2025] [Indexed: 05/29/2025] Open
Abstract
Enhanced hepatic synthesis, assembly, and secretion of apolipoprotein B (ApoB)-containing lipoproteins elevate their plasma levels and-like their impaired clearance from the circulation-can increase cardiovascular risk. Both dietary fatty acids and microRNAs contribute to the nutrient-dependent regulation of hepatic gene expression. Together, these factors may modulate lipid and ApoB-containing lipoprotein synthesis in the liver, either exacerbating or mitigating dyslipidemia. Research continues to reveal the complexity of fatty acid-microRNA networks and highlights differences in regulating hepatic ApoB-containing lipoprotein synthesis between humans and rodents. Consequently, this review focuses on studies conducted in humans or human-derived hepatocytes.
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Affiliation(s)
- Joanna Karbowska
- Department of Biochemistry, Medical University of Gdansk, 80-211 Gdansk, Poland
| | - Zdzislaw Kochan
- Laboratory of Nutritional Biochemistry, Department of Clinical Nutrition, Medical University of Gdansk, 80-211 Gdansk, Poland
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2
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Sun C, Du M, Sha S, Wang S, Li L, Hou J, Li L, Yuan J, Yan J, Yang Z. Puerarin improves MASLD by remodeling intestinal microenvironment to promote mitochondrial fusion and autophagy. J Pharmacol Sci 2025; 158:27-41. [PMID: 40121054 DOI: 10.1016/j.jphs.2025.03.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2024] [Revised: 02/27/2025] [Accepted: 03/03/2025] [Indexed: 03/25/2025] Open
Abstract
Pueraria Mirifica (Willd. Ohwi) is a medicine with anti-inflammatory and lipid-lowering properties. Puerarin is one of the main active components of Pueraria. The aim of this study was to investigate the possible mechanisms of Pueraria in the treatment of non-alcoholic fatty liver disease. The therapeutic effect of the drug was demonstrated by serum and liver pathology indicators. The mechanism of action of puerarin was demonstrated by intestinal microbial changes and short-chain fatty acid content tests. The mechanism of puerarin alleviating Metabolic dysfunction-associated steatotic liver disease (MASLD) by regulating intestinal flora was predicted by bioinformatics. The relationship between flora and liver was revealed by q-PCR detection of mRNA expression level of liver metabolic genes. And the mechanism of puerarin action was further verified by intestinal microbial clearance experiments. Puerarin reduced vacuolar-like changes, lipid deposition, and inflammatory cell infiltration in the livers of high-fat diet (HFD) model mice. The gut microbiota abundance and diversity were remodeled, short-chain fatty acid content was increased, and the intestinal mucosal barrier was repaired, accompanied by a reduction in inflammatory cytokines. Puerarin regulated mRNA expression of hepatic lipid metabolism genes to alleviate MASLD. These results suggested that puerarin treats MASLD by treating altering bacterial metabolism and anti-inflammation.
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Affiliation(s)
- Chunbin Sun
- Yunnan Provincial Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Chinese Medicine, Kunming, 650500, Yunnan, China; School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100080, China
| | - Mei Du
- Guangzhou University of Chinese Medicine, Guangzhou, 510006, China; Department of Oncology, Linyi People's Hospital, Linyi, 276000, China
| | - Shuang Sha
- School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100080, China
| | - Si Wang
- The First School of Clinical Medicine, Yunnan University of Chinese Medicine, Kunming, 650500, China
| | - Lei Li
- Yunnan Provincial Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Chinese Medicine, Kunming, 650500, Yunnan, China
| | - Jiong Hou
- Yunnan Provincial Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Chinese Medicine, Kunming, 650500, Yunnan, China
| | - Li Li
- Yunnan Provincial Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Chinese Medicine, Kunming, 650500, Yunnan, China
| | - Jiali Yuan
- Yunnan Provincial Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Chinese Medicine, Kunming, 650500, Yunnan, China
| | - Jinyuan Yan
- Central Laboratory, Kunming Medical University Second Hospital, Kunming, 650000, Yunnan, China
| | - Zhongshan Yang
- Yunnan Provincial Key Laboratory of Molecular Biology for Sinomedicine, Yunnan University of Chinese Medicine, Kunming, 650500, Yunnan, China.
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3
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Ramandi A, Diehl AM, Sanyal AJ, de Jong YP. Experimental Models to Investigate PNPLA3 in Liver Steatosis. Liver Int 2025; 45:e70091. [PMID: 40231787 DOI: 10.1111/liv.70091] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/14/2025] [Revised: 03/26/2025] [Accepted: 03/30/2025] [Indexed: 04/16/2025]
Abstract
Patatin-like phospholipase domain-containing 3 (PNPLA3) was the first gene identified through genome-wide association studies to be linked to hepatic fat accumulation. A missense variant, encoding the PNPLA3-148M allele, has since been shown to increase the risk for the full spectrum of steatotic liver disease (SLD), from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Despite extensive validation of this association and ongoing research into its pathogenic role, the precise mechanisms by which PNPLA3-148M contributes to the progression of SLD remain poorly understood. In this review, we evaluate preclinical in vitro and in vivo models used to investigate PNPLA3 and its involvement in SLD, with particular emphasis on metabolic dysfunction-associated steatotic liver disease. We assess the strengths and limitations of these models, as well as the challenges arising from species differences in PNPLA3 expression and function between human and murine systems.
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Affiliation(s)
- Alireza Ramandi
- Division of Gastroenterology and Hepatology, Weill Cornell Medicine, New York, New York, USA
| | - Anna-Mae Diehl
- Department of Medicine, Duke University, Durham, North Carolina, USA
| | - Arun J Sanyal
- Stravitz-Sanyal Institute for Liver Disease and Metabolic Health, Virginia Commonwealth University, Richmond, Virginia, USA
| | - Ype P de Jong
- Division of Gastroenterology and Hepatology, Weill Cornell Medicine, New York, New York, USA
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4
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Hawkins C, Waddilove E, Matthews PC, Delphin M. Impact of metformin on HBV replication: No evidence of suppression in vitro. J Clin Virol 2025; 177:105781. [PMID: 40120569 DOI: 10.1016/j.jcv.2025.105781] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2025] [Revised: 03/12/2025] [Accepted: 03/15/2025] [Indexed: 03/25/2025]
Abstract
BACKGROUND Outcomes of chronic Hepatitis B (CHB) infection have been increasingly associated with various metabolic syndromes, including metabolic-dysfunction associated steatotic liver disease (MASLD), with a potential for impact on liver disease progression. There is some evidence that metformin, a widely used anti-diabetic drug, may reduce hepatocellular carcinoma (HCC) incidence in people living with Hepatitis B Virus (HBV), but with little to no evidence of impact on the virus itself in vivo. However, previous in vitro studies suggest metformin may have a direct impact on HBV replication, although the mechanism remains unclear. OBJECTIVES We aimed to investigate the impact of metformin on HBV replication in vitro. STUDY DESIGN Hepatocyte cell lines constitutively expressing HBV (HepAD38) were treated once or thrice with escalating doses of metformin, using lamivudine and water as controls. We monitored cellular cytotoxicity as well as HBV biomarkers (HBeAg, HBsAg, HBV DNA and RNA) throughout the assay. RESULTS We did not observe any impact of metformin on HBV replication after a single dose or three repeated treatments. CONCLUSIONS In HepAD38 cells, HBV replication is not impacted by metformin treatment. This contrasts with prior in vitro data but is in line with clinical evidence that suggests metformin acts through an influence on liver disease progression rather than a direct antiviral impact on HBV itself.
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Affiliation(s)
- Cyrus Hawkins
- The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
| | | | - Philippa C Matthews
- The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK; Division of Infection and Immunity, University College London, Gower Street, London WC1E 6BT, UK; Department of Infectious Diseases, University College London Hospital, Euston Road, London NW1 2BU, UK
| | - Marion Delphin
- The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
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5
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Stilkerich A, Schicht G, Seidemann L, Hänsel R, Friebel A, Hoehme S, Seehofer D, Damm G. Cell Homeostasis or Cell Death-The Balancing Act Between Autophagy and Apoptosis Caused by Steatosis-Induced Endoplasmic Reticulum (ER) Stress. Cells 2025; 14:449. [PMID: 40136698 PMCID: PMC11941029 DOI: 10.3390/cells14060449] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2024] [Revised: 03/04/2025] [Accepted: 03/07/2025] [Indexed: 03/27/2025] Open
Abstract
Metabolic-dysfunction-associated steatotic liver disease (MASLD) is a prevalent liver condition with potential progression to cirrhosis and impaired regeneration post-resection. A key mechanism underlying lipotoxicity is endoplasmic reticulum (ER) stress, particularly the activation of the unfolded protein response (UPR). This study investigates the interplay between lipid accumulation, endoplasmic reticulum (ER) stress, and cellular outcomes, focusing on the balance between autophagy and apoptosis. We cultured primary human hepatocytes (PHH) in a free fatty acid (FFA)-enriched medium for 120 h, assessing lipid accumulation, metabolism, and the expression of selected UPR markers. Additionally, we investigated the effects of lipid load on cell activity and growth in proliferating HepG2 cells. We observed that FFA uptake consistently induced ER stress, shifting cellular responses toward apoptosis under high lipid loads. Donor-specific differences were evident, particularly in lipid storage, excretion, and sensitivity to lipotoxicity. Some donors exhibited limited triglyceride (TAG) storage and excretion, leading to an excess of FFA whose metabolic fate remains unclear. Proliferation was more sensitive to lipid accumulation than overall cell activity, with even low FFA concentrations impairing growth, highlighting the vulnerability of regenerative processes to steatosis. The study elucidates how ER stress pathways, such as PERK-CHOP and IRE1α-JNK, are differentially activated in response to lipid overload, tipping the balance toward apoptosis in severe cases. The limited activation of repair mechanisms, such as autophagy, further emphasizes the critical role of ER stress in determining hepatocyte fate. The donor-dependent variability highlights the need for personalized strategies to mitigate lipotoxic effects and enhance liver regeneration in steatosis-related conditions.
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Affiliation(s)
- Anna Stilkerich
- Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, 04103 Leipzig, Germany; (A.S.); (G.S.); (L.S.); (D.S.)
- Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, 04103 Leipzig, Germany; (R.H.); (A.F.); (S.H.)
| | - Gerda Schicht
- Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, 04103 Leipzig, Germany; (A.S.); (G.S.); (L.S.); (D.S.)
- Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, 04103 Leipzig, Germany; (R.H.); (A.F.); (S.H.)
| | - Lena Seidemann
- Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, 04103 Leipzig, Germany; (A.S.); (G.S.); (L.S.); (D.S.)
- Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, 04103 Leipzig, Germany; (R.H.); (A.F.); (S.H.)
| | - René Hänsel
- Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, 04103 Leipzig, Germany; (R.H.); (A.F.); (S.H.)
| | - Adrian Friebel
- Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, 04103 Leipzig, Germany; (R.H.); (A.F.); (S.H.)
- Interdisciplinary Center for Bioinformatics (IZBI), University of Leipzig, Haertelstraße 16-18, 04107 Leipzig, Germany
- Center for Scalable Data Analytics and Artificial Intelligence (ScaDS.AI), 04105 Leipzig, Germany
| | - Stefan Hoehme
- Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, 04103 Leipzig, Germany; (R.H.); (A.F.); (S.H.)
- Interdisciplinary Center for Bioinformatics (IZBI), University of Leipzig, Haertelstraße 16-18, 04107 Leipzig, Germany
- Center for Scalable Data Analytics and Artificial Intelligence (ScaDS.AI), 04105 Leipzig, Germany
| | - Daniel Seehofer
- Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, 04103 Leipzig, Germany; (A.S.); (G.S.); (L.S.); (D.S.)
- Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, 04103 Leipzig, Germany; (R.H.); (A.F.); (S.H.)
| | - Georg Damm
- Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, 04103 Leipzig, Germany; (A.S.); (G.S.); (L.S.); (D.S.)
- Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, 04103 Leipzig, Germany; (R.H.); (A.F.); (S.H.)
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6
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Eccles-Miller JA, Johnson TD, Baldwin WS. Sexually Dimorphic Effects of CYP2B6 in the Development of Fasting-Mediated Steatosis in Mice: Role of the Oxylipin Products 9-HODE and 9-HOTrE. Biomedicines 2025; 13:295. [PMID: 40002708 PMCID: PMC11853041 DOI: 10.3390/biomedicines13020295] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2024] [Revised: 01/17/2025] [Accepted: 01/19/2025] [Indexed: 02/27/2025] Open
Abstract
Background: Cytochrome P450 2B6 (CYP2B6) is a sexually dimorphic, anti-obesity CYP enzyme responsible for the metabolism of xeno- and endobiotics, including the metabolism of polyunsaturated fatty acids (PUFAs) into 9-hydroxyoctadecadienoic acid (9-HODE) and 9-hydroxyoctadecatrienoic acid (9-HOTrE). However, humanized CYP2B6 transgenic (hCYP2B6-Tg) mice are sensitive to diet-induced hepatic steatosis despite their resistance to obesity. The purpose of this study was to determine if 9-HODE, 9-HOTrE, or other factors contribute to the sexually dimorphic steatosis observed in hCYP2B6-Tg mice. Results: Cyp2b9/10/13-null (Cyp2b-null) mice were injected with either 9-HODE or 9-HOTrE for 2 days and were then subjected to a fasting period of 20 h to induce steatosis. Serum lipids were moderately increased, especially in females, after 9-HODE (triglycerides (TGs), very low-density lipoproteins (VLDLs)) and 9-HOTrE (high-density lipoproteins (HDLs), low-density lipoproteins (LDLs), cholesterol) treatment. No change in hepatic lipids and few changes in hepatic gene expression were observed in mice treated with either oxylipin, suggesting that these oxylipins had minimal to moderate effects. Therefore, to further investigate CYP2B6's role in steatosis, hCYP2B6-Tg and Cyp2b-null mice were subjected to a 20 h fast and compared. Both male and female hCYP2B6-Tg mice exhibited increased steatosis compared to Cyp2b-null mice. Serum cholesterol, triglycerides, HDLs, and VLDLs were increased in hCYP2B6-Tg males. Serum triglycerides and VLDLs were decreased in hCYP2B6-Tg females, suggesting the greater hepatic retention of lipids in females. Hepatic oxylipin profiles revealed eight perturbed oxylipins in female hCYP2B6-Tg mice and only one in males when compared to Cyp2b-null mice. RNA-seq also demonstrated greater effects in females in terms of the number of genes and gene ontology (GO) terms perturbed. There were only a few overlapping GO terms between sexes, and lipid metabolic processes were enriched in hCYP2B6-Tg male mice but were repressed in hCYP2B6-Tg females compared to Cyp2b-nulls. Conclusions: hCYP2B6-Tg mice are sensitive to fasting-mediated steatosis in males and females, although the responses are different. In addition, the oxylipins 9-HODE and 9-HOTrE are unlikely to be the primary cause of CYP2B6's pro-steatotic effects.
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Affiliation(s)
| | | | - William S. Baldwin
- Biological Sciences, Clemson University, Clemson, SC 29634, USA; (J.A.E.-M.); (T.D.J.)
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Srnic N, Westcott F, Caney E, Hodson L. Dietary fat quantity and composition influence hepatic lipid metabolism and metabolic disease risk in humans. Dis Model Mech 2025; 18:dmm050878. [PMID: 39878508 PMCID: PMC11810042 DOI: 10.1242/dmm.050878] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2025] Open
Abstract
The excessive accumulation of intrahepatic triglyceride (IHTG) in the liver is a risk factor for metabolic diseases, including type 2 diabetes and cardiovascular disease. IHTG can excessively accumulate owing to imbalances in the delivery, synthesis, storage and disposal of fat to, in and from the liver. Although obesity is strongly associated with IHTG accumulation, emerging evidence suggests that the composition of dietary fat, in addition to its quantity, plays a role in mediating IHTG accumulation. Evidence from human cross-sectional and interventional studies indicates that diets enriched with saturated fat compared to other fat types and carbohydrates produce divergent effects on IHTG content. However, the mechanistic reasons for these observations remain unknown. Given the challenges of investigating such mechanisms in humans, cellular models are needed that can recapitulate human hepatocyte fatty acid metabolism. Here, we review what is known from human studies about how dietary fat, its quantity and composition contribute to IHTG accumulation. We also explore the effects of fatty acid composition on hepatocellular fat metabolism from data generated in cellular models to help explain the divergences observed in in vivo studies.
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Affiliation(s)
- Nikola Srnic
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford OX3 7LE, UK
| | - Felix Westcott
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford OX3 7LE, UK
| | - Eleanor Caney
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford OX3 7LE, UK
| | - Leanne Hodson
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford OX3 7LE, UK
- Oxford NIHR Biomedical Research Centre, Churchill Hospital, Oxford OX3 7LE, UK
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8
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Singla B. Fanlian Huazhuo Formula: A promising herbal preparation for metabolic liver disease. World J Gastroenterol 2024; 30:4964-4968. [PMID: 39679304 PMCID: PMC11612710 DOI: 10.3748/wjg.v30.i46.4964] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/28/2024] [Revised: 10/17/2024] [Accepted: 11/05/2024] [Indexed: 11/21/2024] Open
Abstract
The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has increased significantly in recent decades and is projected to increase further due to the rising obesity rates. MASLD patients are at higher risk of developing advanced liver diseases "cirrhosis and hepatocellular carcinoma" as well as liver- or cardiovascular-related mortality. Existing lipid-lowering therapies failed to reduce the risk of mortality in these patients. Therefore, there is an urgent need for pharmacotherapies that can control and even reverse this disease. Fanlian Huazhuo Formula (FLHZF) is a combination herbal preparation, and its various individual constituents regulate hepatic lipid metabolism, adipose tissue inflammation, and gut microbiota. Despite, these useful effects, limited information is available on its benefits in diet-induced hepatosteatosis. In this article, we discuss the research findings recently published about the therapeutic effects of FLHZF in suppressing MASLD development and underlying mechanisms. Utilizing a series of in vitro and in vivo experiments, the authors demonstrated for the first time that FLHZF suppresses MASLD in male mice possibly by inhibiting hepatic de novo lipogenesis pathways and reducing hepatocyte death. This study paves the way for future investigations aimed at investigating FLHZF's role in inhibiting lipogenesis particularly using radioactively-labeled glucose and acetate, and governing hepatocyte mitochondrial function, gut microbiome profile, and its effects in other models of MASLD, and female mice.
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Affiliation(s)
- Bhupesh Singla
- Pharmaceutical Sciences, The University of Tennessee Health Science Center, Memphis, TN 38103, United States
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9
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Štancl P, Gršković P, Držaić S, Vičić A, Karlić R, Korać P. RNA-Sequencing Identification of Genes Supporting HepG2 as a Model Cell Line for Hepatocellular Carcinoma or Hepatocytes. Genes (Basel) 2024; 15:1460. [PMID: 39596661 PMCID: PMC11593409 DOI: 10.3390/genes15111460] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2024] [Revised: 10/31/2024] [Accepted: 11/06/2024] [Indexed: 11/29/2024] Open
Abstract
Background/Objectives: Cell lines do not faithfully replicate the authentic transcriptomic condition of the disease under study. The HepG2 cell line is widely used for studying hepatocellular carcinoma (HCC), but not all biological processes and genes exhibit congruent expression patterns between cell lines and the actual disease. The objective of this study is to perform a comparative transcriptomic analysis of the HepG2 cell line, HCC, and primary hepatocytes (PH) in order to identify genes suitable for research in HepG2 as a model for PH or HCC research. Methods: We conducted a differential expression analysis between publicly available data from HCC patients, PH, and HepG2. We examined specific overlaps of differentially expressed genes (DEGs) in a pairwise manner between groups in order to obtain a valuable gene list for studying HCC or PH using different parameter filtering. We looked into the function and druggability of these genes. Conclusions: In total, we identified 397 genes for HepG2 as a valuable HCC model and 421 genes for HepG2 as a valuable PH model, and with more stringent criteria, we derived a smaller list of 40 and 21 genes, respectively. The majority of genes identified as a valuable set for the HCC model are involved in DNA repair and protein degradation mechanisms. This research aims to provide detailed guidance on gene selection for studying diseases like hepatocellular carcinoma, primary hepatocytes, or others using cell lines.
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Affiliation(s)
- Paula Štancl
- Bioinformatics Group, Division of Molecular Biology, Department of Biology, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia; (P.Š.); (S.D.)
| | - Paula Gršković
- Biomedical Research Group, Division of Molecular Biology, Department of Biology, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia;
| | - Sara Držaić
- Bioinformatics Group, Division of Molecular Biology, Department of Biology, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia; (P.Š.); (S.D.)
| | - Ana Vičić
- Department of Obstetrics and Gynecology, Clinical Hospital “Sveti Duh”, 10000 Zagreb, Croatia;
| | - Rosa Karlić
- Bioinformatics Group, Division of Molecular Biology, Department of Biology, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia; (P.Š.); (S.D.)
| | - Petra Korać
- Biomedical Research Group, Division of Molecular Biology, Department of Biology, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia;
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10
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Kim C, Zhu Z, Barbazuk WB, Bacher RL, Vulpe CD. Time-course characterization of whole-transcriptome dynamics of HepG2/C3A spheroids and its toxicological implications. Toxicol Lett 2024; 401:125-138. [PMID: 39368564 DOI: 10.1016/j.toxlet.2024.10.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2024] [Revised: 09/10/2024] [Accepted: 10/02/2024] [Indexed: 10/07/2024]
Abstract
Physiologically relevant in vitro models are a priority in predictive toxicology to replace and/or reduce animal experiments. The compromised toxicant metabolism of many immortalized human liver cell lines grown as monolayers as compared to in vivo metabolism limits their physiological relevance. However, recent efforts to culture liver cells in a 3D environment, such as spheroids, to better mimic the in vivo conditions, may enhance the toxicant metabolism of human liver cell lines. In this study, we characterized the dynamic changes in the transcriptome of HepG2/C3A hepatocarcinoma cell spheroids maintained in a clinostat system (CelVivo) to gain insight into the metabolic capacity of this model as a function of spheroid size and culture time. We assessed morphological changes (size, necrotic core), cell health, and proliferation rate from initial spheroid seeding to 35 days of continuous culture in conjunction with a time-course (0, 3, 7, 10, 14, 21, 28 days) of the transcriptome (TempO-Seq, BioSpyder). The phenotypic characteristics of HepG2/C3A growing in spheroids were comparable to monolayer growth until ∼Day 12 (Day 10-14) when a significant decrease in cell doubling rate was noted which was concurrent with down-regulation of cell proliferation and cell cycle pathways over this time period. Principal component analysis of the transcriptome data suggests that the Day 3, 7, and 10 spheroids are pronouncedly different from the Day 14, 21, and 28 spheroids in support of a biological transition time point during the long-term 3D spheroid cultures. The expression of genes encoding cellular components involved in toxicant metabolism and transport rapidly increased during the early time points of spheroids to peak at Day 7 or Day 10 as compared to monolayer cultures with a gradual decrease in expression with further culture, suggesting the most metabolically responsive time window for exposure studies. Overall, we provide baseline information on the cellular and molecular characterization, with a particular focus on toxicant metabolic capacity dynamics and cell growth, of HepG2/C3A 3D spheroid cultures over time.
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Affiliation(s)
- Chanhee Kim
- Center for Human and Environmental Toxicology, Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States
| | - Zhaohan Zhu
- Department of Biostatistics, University of Florida, Gainesville, FL, United States
| | - W Brad Barbazuk
- Department of Biology, University of Florida, Gainesville, FL, United States; University of Florida Genetics Institute, University of Florida, Gainesville, FL, United States
| | - Rhonda L Bacher
- Department of Biostatistics, University of Florida, Gainesville, FL, United States
| | - Christopher D Vulpe
- Center for Human and Environmental Toxicology, Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States.
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11
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Rada P, Carceller-López E, Hitos AB, Gómez-Santos B, Fernández-Hernández C, Rey E, Pose-Utrilla J, García-Monzón C, González-Rodríguez Á, Sabio G, García A, Aspichueta P, Iglesias T, Valverde ÁM. Protein kinase D2 modulates hepatic insulin sensitivity in male mice. Mol Metab 2024; 90:102045. [PMID: 39401614 PMCID: PMC11535753 DOI: 10.1016/j.molmet.2024.102045] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/07/2024] [Revised: 09/30/2024] [Accepted: 10/07/2024] [Indexed: 10/21/2024] Open
Abstract
OBJECTIVES Protein kinase D (PKD) family is emerging as relevant regulator of metabolic homeostasis. However, the precise role of PKD2 in modulating hepatic insulin signaling has not been fully elucidated and it is the aim of this study. METHODS PKD inhibition was analyzed for insulin signaling in mouse and human hepatocytes. PKD2 was overexpressed in Huh7 hepatocytes and mouse liver, and insulin responses were evaluated. Mice with hepatocyte-specific PKD2 depletion (PKD2ΔHep) and PKD2fl/fl mice were fed a chow (CHD) or high fat diet (HFD) and glucose homeostasis and lipid metabolism were investigated. RESULTS PKD2 silencing enhanced insulin signaling in hepatocytes, an effect also found in primary hepatocytes from PKD2ΔHep mice. Conversely, a constitutively active PKD2 mutant reduced insulin-stimulated AKT phosphorylation. A more in-depth analysis revealed reduced IRS1 serine phosphorylation under basal conditions and increased IRS1 tyrosine phosphorylation in PKD2ΔHep primary hepatocytes upon insulin stimulation and, importantly PKD co-immunoprecipitates with IRS1. In vivo constitutively active PKD2 overexpression resulted in a moderate impairment of glucose homeostasis and reduced insulin signaling in the liver. On the contrary, HFD-fed PKD2ΔHep male mice displayed improved glucose and pyruvate tolerance, as well as higher peripheral insulin tolerance and enhanced hepatic insulin signaling compared to control PKD2fl/fl mice. Despite of a remodeling of hepatic lipid metabolism in HFD-fed PKD2ΔHep mice, similar steatosis grade was found in both genotypes. CONCLUSIONS Results herein have unveiled an unknown role of PKD2 in the control of insulin signaling in the liver at the level of IRS1 and point PKD2 as a therapeutic target for hepatic insulin resistance.
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Affiliation(s)
- Patricia Rada
- Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain.
| | - Elena Carceller-López
- Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain
| | - Ana B Hitos
- Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain
| | - Beatriz Gómez-Santos
- Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country UPV/EHU, Leioa, Spain; BioBizkaia Health Research Institute, Barakaldo, Spain
| | - Constanza Fernández-Hernández
- Centro de Metabolómica y Bioanálisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, Boadilla del Monte, Spain
| | - Esther Rey
- Liver Research Unit, Santa Cristina University Hospital, Instituto de Investigación Sanitaria Princesa, Madrid, Spain
| | - Julia Pose-Utrilla
- Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain
| | - Carmelo García-Monzón
- Liver Research Unit, Santa Cristina University Hospital, Instituto de Investigación Sanitaria Princesa, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Madrid, Spain
| | - Águeda González-Rodríguez
- Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain; Liver Research Unit, Santa Cristina University Hospital, Instituto de Investigación Sanitaria Princesa, Madrid, Spain
| | - Guadalupe Sabio
- Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain; Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain
| | - Antonia García
- Centro de Metabolómica y Bioanálisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, Boadilla del Monte, Spain
| | - Patricia Aspichueta
- Department of Physiology, Faculty of Medicine and Nursing, University of the Basque Country UPV/EHU, Leioa, Spain; BioBizkaia Health Research Institute, Barakaldo, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Madrid, Spain
| | - Teresa Iglesias
- Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain
| | - Ángela M Valverde
- Instituto de Investigaciones Biomédicas Sols-Morreale (IIBM), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain; Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain.
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12
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Wu Y, Foollee A, Chan AY, Hille S, Hauke J, Challis MP, Johnson JL, Yaron TM, Mynard V, Aung OH, Cleofe MAS, Huang C, Lim Kam Sian TCC, Rahbari M, Gallage S, Heikenwalder M, Cantley LC, Schittenhelm RB, Formosa LE, Smith GC, Okun JG, Müller OJ, Rusu PM, Rose AJ. Phosphoproteomics-directed manipulation reveals SEC22B as a hepatocellular signaling node governing metabolic actions of glucagon. Nat Commun 2024; 15:8390. [PMID: 39333498 PMCID: PMC11436942 DOI: 10.1038/s41467-024-52703-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2024] [Accepted: 09/16/2024] [Indexed: 09/29/2024] Open
Abstract
The peptide hormone glucagon is a fundamental metabolic regulator that is also being considered as a pharmacotherapeutic option for obesity and type 2 diabetes. Despite this, we know very little regarding how glucagon exerts its pleiotropic metabolic actions. Given that the liver is a chief site of action, we performed in situ time-resolved liver phosphoproteomics to reveal glucagon signaling nodes. Through pathway analysis of the thousands of phosphopeptides identified, we reveal "membrane trafficking" as a dominant signature with the vesicle trafficking protein SEC22 Homolog B (SEC22B) S137 phosphorylation being a top hit. Hepatocyte-specific loss- and gain-of-function experiments reveal that SEC22B was a key regulator of glycogen, lipid and amino acid metabolism, with SEC22B-S137 phosphorylation playing a major role in glucagon action. Mechanistically, we identify several protein binding partners of SEC22B affected by glucagon, some of which were differentially enriched with SEC22B-S137 phosphorylation. In summary, we demonstrate that phosphorylation of SEC22B is a hepatocellular signaling node mediating the metabolic actions of glucagon and provide a rich resource for future investigations on the biology of glucagon action.
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Affiliation(s)
- Yuqin Wu
- Nutrient Metabolism & Signalling Laboratory, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Victoria, Australia
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Victoria, Australia
| | - Ashish Foollee
- Nutrient Metabolism & Signalling Laboratory, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Victoria, Australia
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Victoria, Australia
| | - Andrea Y Chan
- Nutrient Metabolism & Signalling Laboratory, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Victoria, Australia
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Victoria, Australia
| | - Susanne Hille
- Department of Internal Medicine V, University Hospital of Schleswig-Holstein, Campus Kiel, Germany
- German Center for Cardiovascular Research (DZHK), Partner site Hamburg/Kiel/Lübeck, Kiel, Germany
| | - Jana Hauke
- Division of Inherited Metabolic Diseases, University Children's Hospital, Heidelberg, Germany
| | - Matthew P Challis
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Victoria, Australia
| | - Jared L Johnson
- Meyer Cancer Center, Weill Cornell Medicine, New York, USA
- Department of Cell Biology, Harvard Medical School, Boston, USA
- Dana-Farber Cancer Institute, Harvard Medical School, Boston, USA
| | - Tomer M Yaron
- Meyer Cancer Center, Weill Cornell Medicine, New York, USA
- Englander Institute for Precision Medicine, Institute for Computational Biomedicine, Weill Cornell Medicine, New York, USA
- Columbia University Vagelos College of Physicians and Surgeons, New York, USA
| | - Victoria Mynard
- Nutrient Metabolism & Signalling Laboratory, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Victoria, Australia
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Victoria, Australia
| | - Okka H Aung
- Nutrient Metabolism & Signalling Laboratory, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Victoria, Australia
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Victoria, Australia
| | - Maria Almira S Cleofe
- Nutrient Metabolism & Signalling Laboratory, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Victoria, Australia
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Victoria, Australia
| | - Cheng Huang
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Victoria, Australia
- Monash Proteomics and Metabolomics Platform, Monash University, Victoria, Australia
| | | | - Mohammad Rahbari
- German Cancer Research Center (DKFZ), Division of Chronic Inflammation and Cancer, Im Neuenheimer Feld 280, Heidelberg, Germany
- University Hospital Mannheim, Medical Faculty Mannheim, University of Heidelberg, Department of Surgery, Theodor-Kutzer-Ufer 1-3, Heidelberg, Germany
- University Tuebingen, Faculty of Medicine, Institute for Interdisciplinary Research on Cancer Metabolism and Chronic Inflammation, M3-Research Center for Malignome, Metabolome and Microbiome, Otfried-Müller-Straße 37, Tübingen, Germany
| | - Suchira Gallage
- German Cancer Research Center (DKFZ), Division of Chronic Inflammation and Cancer, Im Neuenheimer Feld 280, Heidelberg, Germany
- University Tuebingen, Faculty of Medicine, Institute for Interdisciplinary Research on Cancer Metabolism and Chronic Inflammation, M3-Research Center for Malignome, Metabolome and Microbiome, Otfried-Müller-Straße 37, Tübingen, Germany
| | - Mathias Heikenwalder
- German Cancer Research Center (DKFZ), Division of Chronic Inflammation and Cancer, Im Neuenheimer Feld 280, Heidelberg, Germany
- University Tuebingen, Faculty of Medicine, Institute for Interdisciplinary Research on Cancer Metabolism and Chronic Inflammation, M3-Research Center for Malignome, Metabolome and Microbiome, Otfried-Müller-Straße 37, Tübingen, Germany
- Cluster of Excellence iFIT (EXC 2180) "Image-Guided and Functionally Instructed Tumor Therapies", Eberhard-Karls University, Tübingen, Germany
| | - Lewis C Cantley
- Meyer Cancer Center, Weill Cornell Medicine, New York, USA
- Department of Cell Biology, Harvard Medical School, Boston, USA
- Dana-Farber Cancer Institute, Harvard Medical School, Boston, USA
| | - Ralf B Schittenhelm
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Victoria, Australia
- Monash Proteomics and Metabolomics Platform, Monash University, Victoria, Australia
| | - Luke E Formosa
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Victoria, Australia
| | - Greg C Smith
- School of Biomedical Sciences, University of New South Wales, Sydney, Australia
| | - Jürgen G Okun
- Division of Inherited Metabolic Diseases, University Children's Hospital, Heidelberg, Germany
| | - Oliver J Müller
- Department of Internal Medicine V, University Hospital of Schleswig-Holstein, Campus Kiel, Germany
- German Center for Cardiovascular Research (DZHK), Partner site Hamburg/Kiel/Lübeck, Kiel, Germany
| | - Patricia M Rusu
- Nutrient Metabolism & Signalling Laboratory, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Victoria, Australia
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Victoria, Australia
| | - Adam J Rose
- Nutrient Metabolism & Signalling Laboratory, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Victoria, Australia.
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Faculty of Medicine, Nursing & Health Sciences, Monash University, Victoria, Australia.
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13
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Valente LC, Bacil GP, Riechelmann-Casarin L, Barbosa GC, Barbisan LF, Romualdo GR. Exploring in vitro modeling in hepatocarcinogenesis research: morphological and molecular features and similarities to the corresponding human disease. Life Sci 2024; 351:122781. [PMID: 38848937 DOI: 10.1016/j.lfs.2024.122781] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2024] [Revised: 04/04/2024] [Accepted: 06/04/2024] [Indexed: 06/09/2024]
Abstract
The hepatocellular carcinoma (HCC) features a remarkable epidemiological burden, ranking as the third most lethal cancer worldwide. As the HCC-related molecular and cellular complexity unfolds as the disease progresses, the use of a myriad of in vitro models available is mandatory in translational preclinical research setups. In this review paper, we will compile cutting-edge information on the in vitro bioassays for HCC research, (A) emphasizing their morphological and molecular parallels with human HCC; (B) delineating the advantages and limitations of their application; and (C) offering perspectives on their prospective applications. While bidimensional (2D) (co) culture setups provide a rapid low-cost strategy for metabolism and drug screening investigations, tridimensional (3D) (co) culture bioassays - including patient-derived protocols as organoids and precision cut slices - surpass some of the 2D strategies limitations, mimicking the complex microarchitecture and cellular and non-cellular microenvironment observed in human HCC. 3D models have become invaluable tools to unveil HCC pathophysiology and targeted therapy. In both setups, the recapitulation of HCC in different etiologies/backgrounds (i.e., viral, fibrosis, and fatty liver) may be considered as a fundamental guide for obtaining translational findings. Therefore, a "multimodel" approach - encompassing the advantages of different in vitro bioassays - is encouraged to circumvent "model-biased" outcomes in preclinical HCC research.
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Affiliation(s)
- Leticia Cardoso Valente
- São Paulo State University (UNESP), Medical School, Botucatu, Experimental Research Unit (UNIPEX), Brazil
| | - Gabriel Prata Bacil
- São Paulo State University (UNESP), Institute of Biosciences, Botucatu, Department of Structural and Functional Biology, Brazil
| | - Luana Riechelmann-Casarin
- São Paulo State University (UNESP), Medical School, Botucatu, Experimental Research Unit (UNIPEX), Brazil
| | | | - Luís Fernando Barbisan
- São Paulo State University (UNESP), Institute of Biosciences, Botucatu, Department of Structural and Functional Biology, Brazil
| | - Guilherme Ribeiro Romualdo
- São Paulo State University (UNESP), Medical School, Botucatu, Experimental Research Unit (UNIPEX), Brazil.
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14
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Bonanini F, Singh M, Yang H, Kurek D, Harms AC, Mardinoglu A, Hankemeier T. A comparison between different human hepatocyte models reveals profound differences in net glucose production, lipid composition and metabolism in vitro. Exp Cell Res 2024; 437:114008. [PMID: 38499143 DOI: 10.1016/j.yexcr.2024.114008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2023] [Revised: 03/12/2024] [Accepted: 03/15/2024] [Indexed: 03/20/2024]
Abstract
Hepatocytes are responsible for maintaining a stable blood glucose concentration during periods of nutrient scarcity. The breakdown of glycogen and de novo synthesis of glucose are crucial metabolic pathways deeply interlinked with lipid metabolism. Alterations in these pathways are often associated with metabolic diseases with serious clinical implications. Studying energy metabolism in human cells is challenging. Primary hepatocytes are still considered the golden standard for in vitro studies and have been instrumental in elucidating key aspects of energy metabolism found in vivo. As a result of several limitations posed by using primary cells, a multitude of alternative hepatocyte cellular models emerged as potential substitutes. Yet, there remains a lack of clarity regarding the precise applications for which these models accurately reflect the metabolic competence of primary hepatocytes. In this study, we compared primary hepatocytes, stem cell-derived hepatocytes, adult donor-derived liver organoids, immortalized Upcyte-hepatocytes and the hepatoma cell line HepG2s in their response to a glucose production challenge. We observed the highest net glucose production in primary hepatocytes, followed by organoids, stem-cell derived hepatocytes, Upcyte-hepatocytes and HepG2s. Glucogenic gene induction was observed in all tested models, as indicated by an increase in G6PC and PCK1 expression. Lipidomic analysis revealed considerable differences across the models, with organoids showing the closest similarity to primary hepatocytes in the common lipidome, comprising 347 lipid species across 19 classes. Changes in lipid profiles as a result of the glucose production challenge showed a variety of, and in some cases opposite, trends when compared to primary hepatocytes.
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Affiliation(s)
| | - Madhulika Singh
- Metabolomics and Analytics Center, Leiden Academic Centre for Drug Research, Leiden University, Netherlands
| | - Hong Yang
- Science for Life Laboratory, KTH - Royal Institute of Technology, Stockholm, Sweden
| | | | - Amy C Harms
- Metabolomics and Analytics Center, Leiden Academic Centre for Drug Research, Leiden University, Netherlands
| | - Adil Mardinoglu
- Science for Life Laboratory, KTH - Royal Institute of Technology, Stockholm, Sweden
| | - Thomas Hankemeier
- Metabolomics and Analytics Center, Leiden Academic Centre for Drug Research, Leiden University, Netherlands.
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15
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Xing C, Kemas A, Mickols E, Klein K, Artursson P, Lauschke VM. The choice of ultra-low attachment plates impacts primary human and primary canine hepatocyte spheroid formation, phenotypes, and function. Biotechnol J 2024; 19:e2300587. [PMID: 38403411 DOI: 10.1002/biot.202300587] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2023] [Revised: 01/11/2024] [Accepted: 01/17/2024] [Indexed: 02/27/2024]
Abstract
Organotypic three-dimensional liver spheroid cultures in which hepatic cells retain their molecular phenotype and functionality have emerged as powerful tools for preclinical drug development. In recent years a multitude of culture systems have been developed; however, a thorough side-by-side benchmarking of the different methods is lacking. Here, we compared the performance of ten different 96- and 384-well microplate types to support spheroid formation and long-term culture. Specifically, we evaluated differences in spheroid formation kinetics, viability, functionality, expression patterns, and their utility for hepatotoxicity assessments using primary human hepatocytes (PHH) and primary canine hepatocytes (PCH). All 96-well plates enabled formation of PHH liver spheroids, albeit with differences between plates in spheroid size, geometry, and reproducibility. Performance of different 384-wells was less consistent. Only 6/10 microplates supported the formation of PCH aggregates. Interestingly, even if PCH aggregates in these six microplates were more loosely packed than PHH spheroids, they maintained their function and were compatible with long-term pharmacological and toxicological assays. Overall, Corning and Biofloat plates showed the best performance in the formation of both human and canine liver spheroids with highest viability, most physiologically relevant phenotypes, superior CYP activity and lowest coefficient of variation in toxicity assays. The presented data constitutes a valuable resource that demonstrates the impacts of current ultra-low attachment plates on liver spheroid metrics and can guide evidence-based plate selection. Combined, these results have important implications for the cross-comparison of different studies and can facilitate the standardization and reproducibility of three-dimensional liver culture experiments.
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Affiliation(s)
- Chen Xing
- Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden
| | - Aurino Kemas
- Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden
| | | | - Kathrin Klein
- Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
- University of Tübingen, Tübingen, Germany
| | - Per Artursson
- Department of Pharmacy, Uppsala University, Uppsala, Sweden
| | - Volker M Lauschke
- Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden
- Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
- University of Tübingen, Tübingen, Germany
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16
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Wang S, Lin X, Zhu C, Dong Y, Guo Y, Xie Z, He X, Ju W, Chen M. Association between nonalcoholic fatty liver disease and increased glucose-to-albumin ratio in adults without diabetes. Front Endocrinol (Lausanne) 2024; 14:1287916. [PMID: 38264288 PMCID: PMC10804880 DOI: 10.3389/fendo.2023.1287916] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/03/2023] [Accepted: 11/22/2023] [Indexed: 01/25/2024] Open
Abstract
Background Nonalcoholic fatty liver disease (NAFLD) affects approximately 30% of individuals globally. Both serum glucose and albumin were demonstrated to be potential markers for the development of NAFLD. We hypothesized that the risk of NAFLD may be proportional to the glucose-to-albumin ratio (GAR). Methods Based on information from the National Health and Nutrition Examination Survey (NHANES) 1999-2018, it was determined that GAR was associated with an increased risk of NAFLD and liver fibrosis utilizing weighted multivariable logistic regression. Participants with a fatty liver index (FLI) over 60 were identified with NAFLD, and those with an NAFLD fibrosis score (NFS) >0.676 with evidence of NAFLD were labeled with advanced hepatic fibrosis (AHF). The liver biopsy was utilized to verify the relationship between GAR and FLD in our center cohort. Mendelian randomization analysis investigated the genetic relationship between GAR and NAFLD. Results Of 15,534 eligible participants, 36.4% of participants were identified as NAFLD without AHF. GAR was positively correlated with the probability of NAFLD following full adjustment for possible variables (OR = 1.53, 95% CI: 1.39-1.67). It was confirmed that patients with NAFLD and AHF had an inferior prognosis. The relationship between GAR and NFS was favorable (R = 0.46, P< 0.0001), and NAFLD patients with a higher GAR tended to develop poor survival. In our center cohort, the association between GAR and NAFLD was verified. Conclusion Among participants without diabetes, greater GAR was linked to higher risks of NAFLD. In addition, NAFLD patients with higher GAR tended to develop liver fibrosis and adverse outcomes.
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Affiliation(s)
- Shuai Wang
- Organ Transplant Center, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Organ Donation and Transplant Immunology, Guangzhou, China
- Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation), Guangzhou, China
| | - Xiaohong Lin
- Department of Breast and Thyroid Surgery, Eastern Hospital of the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Chuchen Zhu
- Organ Transplant Center, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Organ Donation and Transplant Immunology, Guangzhou, China
- Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation), Guangzhou, China
| | - Yuqi Dong
- Organ Transplant Center, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Organ Donation and Transplant Immunology, Guangzhou, China
- Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation), Guangzhou, China
| | - Yiwen Guo
- Organ Transplant Center, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Organ Donation and Transplant Immunology, Guangzhou, China
- Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation), Guangzhou, China
| | - Zhonghao Xie
- Organ Transplant Center, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Organ Donation and Transplant Immunology, Guangzhou, China
- Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation), Guangzhou, China
| | - Xiaoshun He
- Organ Transplant Center, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Organ Donation and Transplant Immunology, Guangzhou, China
- Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation), Guangzhou, China
| | - Weiqiang Ju
- Organ Transplant Center, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Organ Donation and Transplant Immunology, Guangzhou, China
- Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation), Guangzhou, China
| | - Maogen Chen
- Organ Transplant Center, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Organ Donation and Transplant Immunology, Guangzhou, China
- Guangdong Provincial International Cooperation Base of Science and Technology (Organ Transplantation), Guangzhou, China
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Wang Q, Liu J, Yin W, Wang A, Zheng J, Wang Y, Dong J. Microscale tissue engineering of liver lobule models: advancements and applications. Front Bioeng Biotechnol 2023; 11:1303053. [PMID: 38144540 PMCID: PMC10749204 DOI: 10.3389/fbioe.2023.1303053] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2023] [Accepted: 11/28/2023] [Indexed: 12/26/2023] Open
Abstract
The liver, as the body's primary organ for maintaining internal balance, is composed of numerous hexagonal liver lobules, each sharing a uniform architectural framework. These liver lobules serve as the basic structural and functional units of the liver, comprised of central veins, hepatic plates, hepatic sinusoids, and minute bile ducts. Meanwhile, within liver lobules, distinct regions of hepatocytes carry out diverse functions. The in vitro construction of liver lobule models, faithfully replicating their structure and function, holds paramount significance for research in liver development and diseases. Presently, two primary technologies for constructing liver lobule models dominate the field: 3D bioprinting and microfluidic techniques. 3D bioprinting enables precise deposition of cells and biomaterials, while microfluidics facilitates targeted transport of cells or other culture materials to specified locations, effectively managing culture media input and output through micro-pump control, enabling dynamic simulations of liver lobules. In this comprehensive review, we provide an overview of the biomaterials, cells, and manufacturing methods employed by recent researchers in constructing liver lobule models. Our aim is to explore strategies and technologies that closely emulate the authentic structure and function of liver lobules, offering invaluable insights for research into liver diseases, drug screening, drug toxicity assessment, and cell replacement therapy.
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Affiliation(s)
- Qi Wang
- Department of Hepatobiliary and Pancreatic Surgery, The First Hospital of Jilin University, Jilin University, Changchun, China
| | - Juan Liu
- Research Unit of Precision Hepatobiliary Surgery Paradigm, Chinese Academy of Medical Sciences, Beijing, China
- Hepato-Pancreato-Biliary Center, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing, China
- Institute for Organ Transplant and Bionic Medicine, Tsinghua University, Beijing, China
- Key Laboratory of Digital Intelligence Hepatology, Ministry of Education, School of Clinical Medicine, Tsinghua University, Beijing, China
| | - Wenzhen Yin
- Clinical Translational Science Center, Beijing Tsinghua Changgung Hospital, Tsinghua University, Beijing, China
| | - Anqi Wang
- Hepato-Pancreato-Biliary Center, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing, China
| | - Jingjing Zheng
- Hepato-Pancreato-Biliary Center, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing, China
| | - Yunfang Wang
- Research Unit of Precision Hepatobiliary Surgery Paradigm, Chinese Academy of Medical Sciences, Beijing, China
- Hepato-Pancreato-Biliary Center, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing, China
- Institute for Organ Transplant and Bionic Medicine, Tsinghua University, Beijing, China
- Key Laboratory of Digital Intelligence Hepatology, Ministry of Education, School of Clinical Medicine, Tsinghua University, Beijing, China
- Clinical Translational Science Center, Beijing Tsinghua Changgung Hospital, Tsinghua University, Beijing, China
| | - Jiahong Dong
- Department of Hepatobiliary and Pancreatic Surgery, The First Hospital of Jilin University, Jilin University, Changchun, China
- Research Unit of Precision Hepatobiliary Surgery Paradigm, Chinese Academy of Medical Sciences, Beijing, China
- Hepato-Pancreato-Biliary Center, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing, China
- Institute for Organ Transplant and Bionic Medicine, Tsinghua University, Beijing, China
- Key Laboratory of Digital Intelligence Hepatology, Ministry of Education, School of Clinical Medicine, Tsinghua University, Beijing, China
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