Given the obesity epidemic and the prevalence of comorbidities, there is an ongoing need to understand the health consequences of this disease state better. Understanding gene expression signals will facilitate the identification of mechanisms of kidney and liver dysfunction/disease often present in individuals with obesity. qPCR is the standard method for studying changes in relative gene expression. Reference genes (RGs) are obligatory for accurately normalizing mRNA transcript levels across samples. Despite the prevalence of qPCR, the reliability of the data is often compromised because RGs are still used without validation or have proven to be unstable in different tissues and various diseases. In this study, we validated seven reference genes (ACTB, B2M, RPLP0, HPRT1, GAPDH, 18S rRNA, and PPIA) using human liver tissue from 15 lean individuals and 17 individuals with a BMI ≥ 25 and human kidney tissue from 13 lean individuals and 15 individuals with a BMI ≥ 25. Cross-validation of expression stability was performed using the RefFinder platform with four algorithms: NormFinder, BestKeeper, geNorm, and the comparative ΔCt method. In obesity-related studies, the most suitable reference genes in gene expression studies are RPLP0 and HPRT1 in human kidney tissue and RPLP0 and GAPDH in the liver.

This study aimed to determine whether remnant cholesterol (RC) and its variability can predict the onset of metabolic dysfunction-associated steatotic liver disease (MASLD) independently of low-density lipoprotein cholesterol (LDL-C) levels. A longitudinal cohort study involving 43,065 participants who underwent at least two physical examinations was conducted. This study used Cox proportional hazards models to assess the relationships among RC quartile levels (Q1-Q4), visit-to-visit variability, and the risk of MASLD. This variability was quantified using several metrics: standard deviation (SD), logSD, average real variability (ARV), logARV, mean absolute deviation (MAD), and logMAD. Concurrently, this study utilized a combined analysis of RC and LDL-C groups to assess the independent risk of MASLD associated with RC. During a mean visit-to-visit of 3.19 years (SD 2.06 years), 8374 patients (19.45%) developed MASLD. Compared with Q1, Q4 was associated with a significantly greater risk of MASLD (hazard ratio [HR] 1.309, 95% confidence interval [CI] 1.220-1.403, P < 0.001). The fully adjusted Cox model revealed that the HRs of SD, logSD, ARV, logARV, MAD and logMAD were 1.400 (95% CI 1.305-1.502), 1.278 (95% CI 1.188-1.374), 1.152 (95% CI 1.079-1.229), 1.183 (95% CI 1.140-1.227), 1.578 (95% CI 1.433-1.737) and 1.263 (95% CI 1.175-1.358), respectively. In both LDL-C subgroups (≥ 3.4 mmol/L and < 3.4 mmol/L), high baseline RC was associated with elevated MASLD risk (HR 1.208, 95% CI 1.148-1.270, P < 0.001; HR 1.246, 95% CI 1.129-1.374, P < 0.001). RC levels were independently associated with MASLD in healthy individuals, irrespective of LDL-C level. The variability of RC during visit-to-visit periods provides a predictive marker for identifying individuals at heightened risk of MASLD.

Nonalcoholic fatty liver disease (NAFLD) represents an increasing public health concern. The underlying pathophysiological mechanisms of NAFLD remains unclear, and as a result, there is currently no specific therapy for this condition. However, recent studies focus on extracellular vesicles (EVs) as a novelty in their role in cellular communication. An imbalance in the gut microbiota composition may contribute to the progression of NAFLD, making the gut-liver axis a promising target for therapeutic strategies. This review aims to provide a comprehensive overview of EVs in NAFLD. Additionally, exosome-like nanovesicles derived from plants (PELNs) and probiotics-derived extracellular vesicles (postbiotics) have demonstrated the potential to re-establish intestinal equilibrium and modulate gut microbiota, thus offering the potential to alleviate NAFLD via the gut-liver axis. Further research is needed using multiple omics approaches to comprehensively characterize the cargo including protein, metabolites, genetic material packaged, and biological activities of extracellular vesicles derived from diverse microbes and plants.

Inflammaging is a process of cellular dysfunction associated with chronic inflammation, which plays a significant role in the onset and progression of liver diseases. Research on its mechanisms has become a hotspot. In viral hepatitis, inflammaging primarily involve oxidative stress, cell apoptosis and necrosis, as well as gut microbiota dysbiosis. In non-alcoholic fatty liver disease, inflammaging is more complex, involving insulin resistance, fat deposition, lipid metabolism disorders, gut microbiota dysbiosis, and abnormalities in NAD+ metabolism. In liver tumors, inflammaging is characterized by weakening of tumor suppressive mechanisms, remodeling of the liver microenvironment, metabolic reprogramming, and enhanced immune evasion. Therapeutic strategies targeting inflammaging have been developing recently, and antioxidant therapy, metabolic disorder improvement, and immunotherapy are emerging as important interventions for liver diseases. This review focuses on the mechanisms of inflammaging in liver diseases, aiming to provide novel insights for the prevention and treatment of liver diseases.

The aim of this study was to investigate the protective effects and potential mechanisms of Tetrahydrocurcumin (THC) on methionine-choline-deficient diet (MCD)-induced MASH in C57BL/6 mice by using multi-omics techniques. The C57BL/6 mice were fed with the MCD for 8 weeks to establish a MASH model, while THC (100 mg·kg-1·d-1) and obeticholic acid (6.5 mg·kg-1·d-1) were administered via gavage to the THC group and the positive control group, respectively. The biochemical indexes of the serum and liver were detected using kits. Liver tissue sections were taken to observe the pathomorphological changes. Serum lipid and bile acid contents were measured via LC-MS, and the changes in ileal intestinal flora were detected by 16S rDNA high-throughput sequencing technology. The results revealed that THC significantly attenuated oxidative stress and lipid accumulation in NCTC-1469 cells and relieved hepatic injury and oxidative stress, reduced hepatic TG content, and improved hepatic steatosis in mice. THC alleviated 34 lipid abnormalities caused by the MCD; increased the abundance and diversity of intestinal flora, the ratio of Firmicutes to Bacteroidota, and the abundance of the probiotic (Verrucomicrobiota, Christensenellaceae, Akkermansiaceae, Lachnospiraceae, Desulfovibrionaceae); and reduced the abundance of obesity-associated pathogenic flora such as Firmicutes. Bile acid analysis showed that THC administration reduced the levels of serum toxic bile acid 7-KDCA and CA. In addition, RT-qPCR studies showed that THC down-regulated the transcript levels of the hepatic lipogenesis-related genes Srebp1c, Acc1, Scd1, and Fas, and up-regulated the transcript levels of the hepatic bile acid secretion-related genes Mrp2 and Bsep. The above results suggest that THC may alleviate MCD-induced MASH by downregulating liver Srebp1c, Acc1, Scd1, and Fas levels to inhibit lipid synthesis, upregulating Mrp2 and Bsep levels to regulate serum toxic BA levels, up-regulating the abundance of intestinal probiotic flora, and down-regulating the abundance of intestinal harmful bacterial flora. The multi-omics findings from the above study identified potential new mechanisms by which THC alleviates MASH, providing new reference targets for the development of anti-MASH drugs. These results also offer a basis for screening clinical diagnostic biomarkers for MASH and provide new directions for personalized diagnosis and treatment.

Non-alcoholic fatty liver disease (NAFLD), now recognized as metabolic dysfunction-associated steatotic liver disease (MASLD), represents a significant and escalating global health challenge. Its prevalence is intricately linked to obesity, insulin resistance, and other components of the metabolic syndrome. As our comprehension of MASLD deepens, it has become evident that this condition extends beyond the liver, embodying a complex, multi-systemic disease with hepatic manifestations that mirror the broader metabolic landscape. This comprehensive review delves into the critical interplay between the gut-liver axis and oxidative stress, elucidating their pivotal roles in the etiology and progression of MASLD. Our analysis reveals several key findings: (1) Bile acid dysregulation can trigger oxidative stress through enhanced ROS production in hepatocytes and Kupffer cells, leading to mitochondrial dysfunction and lipid peroxidation; (2) Gut microbiota dysbiosis disrupts intestinal barrier function, allowing increased translocation of endotoxins like LPS, which activate inflammatory pathways through TLR4 signaling and promote oxidative stress via NADPH oxidase activation; (3) The redox-sensitive transcription factors NF-κB and Nrf2 serve as crucial mediators in the gut-liver axis, with NF-κB regulating inflammatory responses and Nrf2 orchestrating antioxidant defenses; (4) Oxidative stress-induced damage to intestinal barrier function creates a destructive feedback loop, further exacerbating liver inflammation and disease progression. These findings highlight the complex interrelationship between gut-liver axis dysfunction and oxidative stress in MASLD pathogenesis, suggesting potential therapeutic targets for disease management.

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a spectrum of liver lesions, ranging from simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH), that may further progress to cirrhosis. MASLD is estimated to affect more than one third of the general population and it represents a risk factor for end-stage liver failure and liver cancer, substantially contributing to liver-related morbidity and mortality. Although the pathogenesis of MASLD is incompletely understood, it is known to consist of a multifactorial process influenced by extrinsic and intrinsic factors such as metabolic, environmental and demographic features, gut microbiota and genetics. Dysregulation of both extracellular and intracellular lipid composition is known to promote the generation of toxic lipid species, thereby triggering lipotoxicity and cellular stress. These events ultimately lead to the activation of distinct cell death pathways, resulting in inflammation, fibrogenesis and, eventually, carcinogenesis. In this manuscript, we provide a comprehensive review of the role of lipotoxicity during MASLD pathogenesis, discussing the most relevant lipid species and related molecular mechanisms, summarizing the cell type-specific effects and highlighting the most promising putative therapeutic strategies for modulating lipotoxicity and lipid metabolism in MASLD.

Long-chain free fatty acids (FFAs) accumulation and oxidative toxicity is a major cause for several pathological conditions. The mechanisms underlying FFA cytotoxicity remain elusive. Here we show that palmitic acid (PA), the most abundant FFA in the circulation, induces S403 phosphorylation of SQSTM1/p62 (sequestosome 1) and its aggregation, which sequesters KEAP1 and activates the non-canonical SQSTM1-KEAP1-NFE2L2 antioxidant pathway. The PA-induced SQSTM1 S403 phosphorylation and aggregation are dependent on SQSTM1 K7-D69 hydrogen bond formation and dimerization in the Phox and Bem1 (PB1) domain, which facilitates the recruitment of TBK1 that phosphorylates SQSTM1 S403. The ubiquitin E3 ligase TRIM21 ubiquitinates SQSTM1 at the K7 residue and abolishes the PB1 dimerization, S403 phosphorylation, and SQSTM1 aggregation. TRIM21 is oxidized at C92, C111, and C114 to form disulfide bonds that lead to its oligomerization and decreased E3 activity. Mutagenizing the three C residues to S (3CS) abolishes TRIM21 oligomerization and increases its E3 activity. TRIM21 ablation leads to decreased SQSTM1 K7 ubiquitination, hence elevated SQSTM1 S403 phosphorylation and aggregation, which confers protection against PA-induced oxidative stress and cytotoxicity. Therefore, TRIM21 is a negative regulator of SQSTM1 phosphorylation, aggregation, and the antioxidant sequestration function. TRIM21 is oxidized to reduce its E3 activity that helps enhance the SQSTM1-KEAP1-NFE2L2 antioxidant pathway. Inhibition of TRIM21 May be a viable strategy to protect tissues from lipotoxicity resulting from long-chain FFAs.Abbreviations: ER: endoplasmic reticulum; FFA: free fatty acid; HMOX1/HO-1: heme oxygenase 1; IB: immunoblotting; IF: immunofluorescence; IP: immunoprecipitation; KEAP1: kelch like ECH associated protein 1; MASH: metabolic dysfunction-associated steatohepatitis; MEF: mouse embryonic fibroblast; NFE2L2/Nrf2: NFE2 like BZIP transcription factor 2; PA: palmitic acid; PB1: Phox and Bem 1; ROS: reactive oxygen species; SLD: steatotic liver disease; SQSTM1: sequestosome 1; TBK1: TANK-binding kinase 1; TRIM21: tripartite motif containing 21.

The incidence of type 2 diabetes mellitus (T2DM), a major lifestyle-related disease, is increasing worldwide. T2DM, which accounts for approximately 90-95% of all diabetes mellitus cases, is caused by deficient insulin secretion, tissue insulin resistance, or both. Many therapeutic drugs for T2DM have been developed that target the pancreas, which secretes insulin. The liver is the central organ for glucose and lipid metabolism, and failure of hepatic regulatory mechanisms leads to hyperglycemia, insulin resistance, and lipid accumulation. Here, we focused on the liver as a novel therapeutic target for T2DM. The fatty acid composition of phospholipids, a major component of biological membranes, has received considerable research attention owing to their involvement in T2DM onset and progression. Fatty acids in phospholipids are cleaved by phospholipase A to form lysophospholipids, which are subsequently remodeled back into phospholipids by lysophospholipid acyltransferases (LPLATs). LPLATs play an important role in lipid metabolism and homeostasis by regulating the abundance of various phospholipid species in multiple cell and tissue types. We investigated whether overexpression of LPLAT10, also called LPCAT4 and LPEAT2, in the liver could improve abnormalities in glucose metabolism and help treat T2DM. For overexpression, we generated an LPLAT10-expressing adenovirus (Ad) vector using an improved Ad vector named Ad-E4-122aT, which exhibited higher and longer-term transgene expression and lower hepatotoxicity than conventional Ad vectors. In this article, we review the current findings that changes in hepatic phospholipid species due to liver-specific LPLAT10 overexpression affect the pancreas and suppress postprandial hyperglycemia by increasing postprandial insulin secretion.

This is the summary report of the 5th Translational Hepatology Meeting, endorsed by the Spanish Association for the Study of the Liver (AEEH) and held in Seville, Spain, in October 2023. The meeting aimed to provide an update on the latest advances in the field of basic and translational hepatology, covering different molecular, cellular, and pathophysiological aspects of the most relevant clinical challenges in liver pathologies. This includes the identification of novel biomarkers and diagnostic tools, the understanding of the relevance of immune response and inflammation in liver diseases, the characterization of current medical approaches to reverse liver diseases, the incorporation of novel molecular insights through omics techniques, or the characterization of the impact of toxic and metabolic insults, as well as other organ crosstalk, in liver pathophysiology.

Cell membranes consist of heterogeneous lipid nanodomains that influence key cellular processes. Using FRET-based fluorescent assays and fluorescence lifetime imaging microscopy (FLIM), we find that the dimension of cholesterol-enriched ordered membrane domains (OMD) varies considerably, depending on specific cell types. Particularly, nociceptor dorsal root ganglion (DRG) neurons exhibit large OMDs. Disruption of OMDs potentiated action potential firing in nociceptor DRG neurons and facilitated the opening of native hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels. This increased neuronal firing is partially due to an increased open probability and altered gating kinetics of HCN channels. The gating effect on HCN channels is likely due to a direct modulation of their voltage sensors by OMDs. In animal models of neuropathic pain, we observe reduced OMD size and a loss of HCN channel localization within OMDs. Additionally, cholesterol supplementation inhibited HCN channels and reduced neuronal hyperexcitability in pain models. These findings suggest that disturbances in lipid nanodomains play a critical role in regulating HCN channels within nociceptor DRG neurons, influencing pain modulation.

Fatty acid-binding protein 1 (FABP1) plays an important role in regulating fatty acid metabolism in liver, which is a potential therapeutic target for diseases such as non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanisms are not well defined. Using complementary experimental models, we discovered FABP1 induction in hepatocytes as a primary mediator of lipogenesis when exposed to fatty acids, especially saturated fatty acids (SFAs). In the feeding trial, palm oil led to excess lipid accumulation in the liver of large yellow croaker (Larimichthys crocea), accompanied by significant induction of FABP1. In cultured cells, palmitic acid (PA), a kind of SFA, triggered the fabp1 expression and increased triglyceride (TG) contents. Knockdown of FABP1 dampened PA-induced TG accumulation through mitigated lipogenesis. The overexpression of FABP1 showed the opposite result. Furthermore, the inactivation of FABP1 led to induction in insulin-induced gene 1 (INSIG1) expression, which attenuated the processing of sterol regulatory element-binding protein 1 (SREBP1) by down-regulating the nuclear-localized SREBP1. These results revealed a previously unrecognized function of FABP1 in response to PA, providing additional evidence for targeting FABP1 in the treatment of NAFLD caused by SFA.

Cell membranes consist of heterogeneous lipid nanodomains that influence key cellular processes. Using FRET-based fluorescent assays and fluorescence lifetime imaging microscopy (FLIM), we found that the dimension of cholesterol-enriched ordered membrane domains (OMD) varies considerably, depending on specific cell types. Particularly, nociceptor dorsal root ganglion (DRG) neurons exhibit large OMDs. Disruption of OMDs potentiated action potential firing in nociceptor DRG neurons and facilitated the opening of native hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels. This increased neuronal firing is partially due to an increased open probability and altered gating kinetics of HCN channels. The gating effect on HCN channels was likely due to a direct modulation of their voltage sensors by OMDs. In animal models of neuropathic pain, we observed reduced OMD size and a loss of HCN channel localization within OMDs. Additionally, cholesterol supplementation inhibited HCN channels and reduced neuronal hyperexcitability in pain models. These findings suggest that disturbances in lipid nanodomains play a critical role in regulating HCN channels within nociceptor DRG neurons, influencing pain modulation.

Nuclear receptor subfamily 2 group E member 1 (Nr2e1) has been regarded as an essential regulator in neural stem cells. However, its function is still not clear in hepatocytes. This study aimed to clarify the effects of Nr2e1-deficiency in hepatocytes in lipotoxic conditions.

Nr2e1-knockdown AML12 ​cells were generated by lentiviral vector transfection. The influences of Nr2e1-deficiency on hepatocyte survival were determined by cell cycle progression and cell apoptosis rate using flow cytometry. Real-time quantitative PCR and Western blot were used to examine the genes and protein expression related to apoptosis, lipid metabolism, and oxidative stress. Meanwhile, RNA sequencing was adopted in liver samples from Nr2e1-knockout (Nr2e1-KO) mice.

Nr2e1 expression was observed with a significant decrease in AML12 ​cells after palmitic acid-stimulation. Knockdown of Nr2e1 in AML12 ​cells resulted in increased sensitivity to lipotoxicity, evidenced by a partial G0/G1 cell-cycle arrest and higher rates of cell apoptosis. Moreover, Nr2e1-knockdown AML12 ​cells presented increased gene expressions relative to lipid synthesis but decreased levels of β-oxidation related genes. Lack of Nr2e1 augmented palmitate-induced oxidative stress in hepatocytes. In vivo, differential genes in Nr2e1-KO mice liver were enriched in pathways associated with liver regeneration and cell proliferation.

This study indicated that hepatocytes lacking Nr2e1 were more susceptible to lipotoxic-mediated damage. Nr2e1 may serve as a potential target for the development of novel therapies for lipotoxicity-induced liver injury.

Hepatocellular carcinoma (HCC) typically develops as a consequence of liver cirrhosis, but HCC epidemiology has evolved drastically in recent years. Metabolic dysfunction-associated steatotic liver disease (MASLD), including metabolic dysfunction-associated steatohepatitis, has emerged as the most common chronic liver disease worldwide and a leading cause of HCC. A substantial proportion of MASLD-associated HCC (MASLD-HCC) also can develop in patients without cirrhosis. The specific pathways that trigger carcinogenesis in this context are not elucidated completely, and recommendations for HCC surveillance in MASLD patients are challenging. In the era of precision medicine, it is critical to understand the processes that define the profiles of patients at increased risk of HCC in the MASLD setting, including cardiometabolic risk factors and the molecular targets that could be tackled effectively. Ideally, defining categories that encompass key pathophysiological features, associated with tailored diagnostic and treatment strategies, should facilitate the identification of specific MASLD-HCC phenotypes. In this review, we discuss MASLD-HCC, including its epidemiology and health care burden, the mechanistic data promoting MASLD, metabolic dysfunction-associated steatohepatitis, and MASLD-HCC. Its natural history, prognosis, and treatment are addressed specifically, as the role of metabolic phenotypes of MASLD-HCC as a potential strategy for risk stratification. The challenges in identifying high-risk patients and screening strategies also are discussed, as well as the potential approaches for MASLD-HCC prevention and treatment.

Metabolic dysfunction-associated steatotic liver disease (MASLD) comprises a spectrum of liver diseases that span simple steatosis, metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis and may progress to cirrhosis and cancer. The pathogenesis of MASLD is multifactorial and is driven by environmental, genetic, metabolic and immune factors. This review will focus on the role of the type 3 cytokines IL-17 and IL-22 in MASLD pathogenesis and progression. IL-17 and IL-22 are produced by similar adaptive and innate immune cells such as Th17 and innate lymphoid cells, respectively. IL-17-related signaling is upregulated during MASLD resulting in increased chemokines and proinflammatory cytokines in the liver microenvironment, enhanced recruitment of myeloid cells and T cells leading to exacerbation of inflammation and liver disease progression. IL-17 may also act directly by activating hepatic stellate cells resulting in increased fibrosis. In contrast, IL-22 is a pleiotropic cytokine with a dominantly protective signature in MASLD and is currently being tested as a therapeutic strategy. IL-22 also exhibits beneficial metabolic effects and abrogates MASH-related inflammation and fibrosis development via inducing the production of anti-oxidants and anti-apoptotic factors. A sex-dependent effect has been attributed to both cytokines, most importantly to IL-22 in MASLD or related conditions. Altogether, IL-17 and IL-22 are key effectors in MASLD pathogenesis and progression. We will review the role of these two cytokines and cells that produce them in the development of MASLD, their interaction with host factors driving MASLD including sexual dimorphism, and their potential therapeutic benefits.

Non-alcoholic fatty liver disease (NAFLD) remains a prevailing etiological agent behind hepatocyte diseases like chronic liver disease. The spectrum of processes involved in NAFLD stages includes hepatic steatosis, non-alcoholic fatty liver, and non-alcoholic steatohepatitis (NASH). Without intervention, the progression of NASH can further deteriorate into cirrhosis and ultimately, hepatocellular carcinoma. The cardinal features that characterize NAFLD are insulin resistance, lipogenesis, oxidative stress and inflammation, extracellular matrix deposition and fibrosis. Due to its complex pathogenesis, existing pharmaceutical agents fail to take a curative or ameliorative effect on NAFLD. Consequently, it is imperative to identify novel therapeutic targets and strategies for NAFLD, ideally to improve the aforementioned key features in patients. As an enterohepatic regulator of bile acid homeostasis, lipid metabolism, and inflammation, FarnesoidX receptor (FXR) is an important pharmacological target for the treatment of NAFLD. Manipulating FXR to regulate lipid metabolic signaling pathways is a potential mechanism to mitigate NAFLD. Therefore, elucidating the modulatory character of FXR in regulating lipid metabolism in NAFLD has the potential to yield groundbreaking perspectives for drug design. This review details recent advances in the regulation of lipid depletion in hepatocytes and investigates the pivotal function of FXR in the progress of NAFLD.

Non-alcoholic steatohepatitis (NASH) is rapidly surpassing viral hepatitis as the primary cause of hepatocellular carcinoma (HCC). However, understanding of NASH-progressed HCC remains poor, which might impede HCC diagnosis and therapy. In this study, we aim to identify shared transcriptional changes between NASH and HCC, of which we focused on E3 ligase TRIM45. We found TRIM45 exacerbates HCC cells proliferation and metastasis in vitro and in vivo. Further transcriptome analysis revealed TRIM45 predominantly affects fatty acid metabolism and oleic acid restored impaired proliferation and metastasis of TRIM45-deficient HCC cells. IP-tandem mass spectrum and FABP5 depriving experiment indicated that TRIM45 enhance fatty acid synthesis depending on FABP5 presence. Interestingly, we found TRIM45 directly added K33-type and K63-type poly-ubiquitin chains to FABP5 NLS domain, which ultimately promoted FABP5 nuclear translocation. Nuclear FABP5 interacted with PPARγ to facilitate downstream lipid synthesis gene expression. We observed TRIM45 accelerated NASH-to-HCC transition and exacerbated both NASH and NASH-HCC with the enhanced fatty acid production in vivo. Moreover, high concentration of fatty acid increased TRIM45 expression. The established mechanism was substantiated by gene expression correlation in TCGA-LIHC. Collectively, our research revealed a common lipid reprograming process in NASH and HCC and identified the cyclical amplification of the TRIM45-FABP5-PPARγ-fatty acid axis. This signaling pathway offers potential therapeutic targets for therapeutic intervention in NASH and NASH-progressed HCC.

The epidemiological burden of liver steatosis associated with metabolic diseases is continuously growing worldwide and in all age classes. This condition generates possible progression of liver damage (i.e., inflammation, fibrosis, cirrhosis, hepatocellular carcinoma) but also independently increases the risk of cardio-metabolic diseases and cancer. In recent years, the terminological evolution from "nonalcoholic fatty liver disease" (NAFLD) to "metabolic dysfunction-associated fatty liver disease" (MAFLD) and, finally, "metabolic dysfunction-associated steatotic liver disease" (MASLD) has been paralleled by increased knowledge of mechanisms linking local (i.e., hepatic) and systemic pathogenic pathways. As a consequence, the need for an appropriate classification of individual phenotypes has been oriented to the investigation of innovative therapeutic tools. Besides the well-known role for lifestyle change, a number of pharmacological approaches have been explored, ranging from antidiabetic drugs to agonists acting on the gut-liver axis and at a systemic level (mainly farnesoid X receptor (FXR) agonists, PPAR agonists, thyroid hormone receptor agonists), anti-fibrotic and anti-inflammatory agents. The intrinsically complex pathophysiological history of MASLD makes the selection of a single effective treatment a major challenge, so far. In this evolving scenario, the cooperation between different stakeholders (including subjects at risk, health professionals, and pharmaceutical industries) could significantly improve the management of disease and the implementation of primary and secondary prevention measures. The high healthcare burden associated with MASLD makes the search for new, effective, and safe drugs a major pressing need, together with an accurate characterization of individual phenotypes. Recent and promising advances indicate that we may soon enter the era of precise and personalized therapy for MASLD/MASH.

Research on retinoid-based cancer prevention, spurred by the effects of vitamin A deficiency on gastric cancer and subsequent clinical studies on digestive tract cancer, unveils novel avenues for chemoprevention. Acyclic retinoids like 4,5-didehydrogeranylgeranoic acid (4,5-didehydroGGA) have emerged as potent agents against hepatocellular carcinoma (HCC), distinct from natural retinoids such as all-trans retinoic acid (ATRA). Mechanistic studies reveal GGA's unique induction of pyroptosis, a rapid cell death pathway, in HCC cells. GGA triggers mitochondrial superoxide hyperproduction and ER stress responses through Toll-like receptor 4 (TLR4) signaling and modulates autophagy, ultimately activating pyroptotic cell death in HCC cells. Unlike ATRA-induced apoptosis, GGA and palmitic acid (PA) induce pyroptosis, underscoring their distinct mechanisms. While all three fatty acids evoke mitochondrial dysfunction and ER stress responses, GGA and PA inhibit autophagy, leading to incomplete autophagic responses and pyroptosis, whereas ATRA promotes autophagic flux. In vivo experiments demonstrate GGA's potential as an anti-oncometabolite, inducing cell death selectively in tumor cells and thus suppressing liver cancer development. This review provides a comprehensive overview of the molecular mechanisms underlying GGA's anti-HCC effects and underscores its promising role in cancer prevention, highlighting its importance in HCC prevention.

Mutations in the gene MTARC1 (mitochondrial amidoxime-reducing component 1) protect carriers from metabolic dysfunction-associated steatohepatitis (MASH) and cirrhosis. MTARC1 encodes the mARC1 enzyme, which is localized to the mitochondria and has no known MASH-relevant molecular function. Our studies aimed to expand on the published human genetic mARC1 data and to observe the molecular effects of mARC1 modulation in preclinical MASH models.

We identified a novel human structural variant deletion in MTARC1, which is associated with various biomarkers of liver health, including alanine aminotransferase levels. Phenome-wide Mendelian Randomization analyses additionally identified novel putatively causal associations between MTARC1 expression, and esophageal varices and cardiorespiratory traits. We observed that protective MTARC1 variants decreased protein accumulation in in vitro overexpression systems and used genetic tools to study mARC1 depletion in relevant human and mouse systems. Hepatocyte mARC1 knockdown in murine MASH models reduced body weight, liver steatosis, oxidative stress, cell death, and fibrogenesis markers. mARC1 siRNA treatment and overexpression modulated lipid accumulation and cell death consistently in primary human hepatocytes, hepatocyte cell lines, and primary human adipocytes. mARC1 depletion affected the accumulation of distinct lipid species and the expression of inflammatory and mitochondrial pathway genes/proteins in both in vitro and in vivo models.

Depleting hepatocyte mARC1 improved metabolic dysfunction-associated steatotic liver disease-related outcomes. Given the functional role of mARC1 in human adipocyte lipid accumulation, systemic targeting of mARC1 should be considered when designing mARC1 therapies. Our data point to plasma lipid biomarkers predictive of mARC1 abundance, such as Ceramide 22:1. We propose future areas of study to describe the precise molecular function of mARC1, including lipid trafficking and subcellular location within or around the mitochondria and endoplasmic reticulum.

Phospholipids (PLs) are principle constituents of biofilms, with their fatty acyl chain composition significantly impacting the biophysical properties of membranes, thereby influencing biological processes. Recent studies have elucidated that fatty acyl chains, under the enzymatic action of lyso‑phosphatidyl‑choline acyltransferases (LPCATs), expedite incorporation into the sn‑2 site of phosphatidyl‑choline (PC), profoundly affecting pathophysiology. Accumulating evidence suggests that alterations in LPCAT activity are implicated in various diseases, including non‑alcoholic fatty liver disease (NAFLD), hepatitis C, atherosclerosis and cancer. Specifically, LPCAT3 is instrumental in maintaining systemic lipid homeostasis through its roles in hepatic lipogenesis, intestinal lipid absorption and lipoprotein secretion. The liver X receptor (LXR), pivotal in lipid homeostasis, modulates cholesterol, fatty acid (FA) and PL metabolism. LXR's capacity to modify PL composition in response to cellular sterol fluctuations is a vital mechanism for protecting biofilms against lipid stress. Concurrently, LXR activation enhances LPCAT3 expression on cell membranes and elevates polyunsaturated PL levels. This activation can ameliorate saturated free FA effects in vitro or endoplasmic reticulum stress in vivo due to lipid accumulation in hepatic cells. Pharmacological interventions targeting LXR, LPCAT and membrane PL components could offer novel therapeutic directions for NAFLD management. The present review primarily focused on recent advancements in understanding the LPCAT3 signaling pathway's role in lipid metabolism related to NAFLD, aiming to identify new treatment targets for the disease.

Leucine is involved in promoting fatty acid oxidation and lipolysis, mediating lipid metabolism and energy homeostasis, thus it has been widely used in livestock production. However, the effects of leucine on fat deposition and nutrition in Shaziling pigs remain unclear. A total of 72 Shaziling pigs (150 days old, weight 35.00 ± 1.00 kg) were randomly divided into 2 groups and fed with basal diet (control group) or basal diet containing 1% leucine (leucine group) for 60 days. The results showed that leucine significantly increased the average daily feed intake but decreased the ratio of feed to gain (P < 0.05), increased the loin muscle area and serum glucose content (P < 0.05) of Shaziling pigs. Besides, leucine regulated the re-distribution of fatty acids from adipose tissue to muscle as it significantly increased the contents of C18:1n-9 and C22:6n-3 (DHA) in the longissimus thoracis while decreased the contents of C22:5n-3 (DPA), C20:5n-3 (EPA), and DHA in the adipose tissue of Shaziling pigs (P < 0.05). Lipidomic analysis showed that the contents of phosphatidylethanolamines (PEs), cardiolipins (CLs), and phosphatidylglycerols (PGs) in the longissimus thoracis and the contents of lysophosphatidylethanolamines (LPEs), ceramides (Cers), phosphatidylinositols (PIs) in adipose tissue of Shaziling pigs were decreased in leucine group (P < 0.05). Collectively, this study clarified that dietary addition of 1% leucine have a better effect on growth performance and the deposition of beneficial fatty acids in the muscle of Shaziling pigs, which is conductive to the production of high quality and healthy pork. In addition, leucine altered the lipid composition of muscle and fat in Shaziling pigs. The related results provide a theoretical basis and application guidance for regulating fat deposition in Shaziling pigs, which is important for the healthy breeding of Shaziling pigs.

A common challenge in microfluidic cell cultures has to do with analysis of cell function without replacing a significant fraction of the culture volume and disturbing local concentration gradients of signals. To address this challenge, we developed a microfluidic cell culture device with an integrated bioanalysis unit to enable on-chip analysis of picoliter volumes of cell-conditioned media. The culture module consisted of an array of 140 microwells with a diameter of 300 m which were made low-binding to promote organization of cells into 3D spheroids. The bioanalysis module contained a droplet generator unit, 15 micromechanical valves and reservoirs loaded with reagents. Each 0.8 nL droplet contained an aliquot of conditioned media mixed with assay reagents. The use of microvalves allowed us to load enzymatic assay and immunoassay into sequentially generated droplets for detection of glucose and albumin, respectively. As a biological application of the microfluidic device, we evaluated hormonal stimulation and glucose consumption of hepatic spheroids. To mimic physiological processes occurring during feeding and fasting, hepatic spheroids were exposed to pancreatic hormones, insulin or glucagon. The droplet-based bioanalysis module was used to measure uptake or release of glucose upon hormonal stimulation. In the future, we intend to use this microfluidic device to mimic and measure pathophysiological processes associated with hepatic insulin resistance and diabetes in the context of metabolic syndrome.

Fatty acids stored in triacylglycerol-rich lipid droplets are assembled with a surface monolayer composed primarily of phosphatidylcholine (PC). Fatty acids stimulate PC synthesis by translocating CTP:phosphocholine cytidylyltransferase (CCT) α to the inner nuclear membrane, nuclear lipid droplets (nLD) and lipid associated promyelocytic leukemia (PML) structures (LAPS). Huh7 cells were used to identify how CCTα translocation onto these nuclear structures are regulated by fatty acids and phosphorylation of its serine-rich P-domain. Oleate treatment of Huh7 cells increased nLDs and LAPS that became progressively enriched in CCTα. In cells expressing the phosphatidic acid phosphatase Lipin1α or 1β, the expanded pool of nLDs and LAPS had a proportional increase in associated CCTα. In contrast, palmitate induced few nLDs and LAPS and inhibited the oleate-dependent translocation of CCTα without affecting total nLDs. Phospho-memetic or phospho-null mutations in the P-domain revealed that a 70% phosphorylation threshold, rather than site-specific phosphorylation, regulated CCTα association with nLDs and LAPS. In vitro candidate kinase and inhibitor studies in Huh7 cells identified cyclin-dependent kinase (CDK) 1 and 2 as putative P-domain kinases. In conclusion, CCTα translocation onto nLDs and LAPS is dependent on available surface area and fatty acid composition, as well as threshold phosphorylation of the P-domain potentially involving CDKs.

The global prevalence and incidence of non-alcoholic fatty liver disease (NAFLD) are exhibiting an increasing trend. NAFLD is characterized by a significant accumulation of lipids, though its underlying mechanism is still unknown. Here we report that high-fat diet (HFD) feeding induced hepatic steatosis in mice, which was accompanied by a reduction in the expression and function of hepatic TRPV2. Moreover, conditional knockout of TRPV2 in hepatocytes exacerbated HFD-induced hepatic steatosis. In an in vitro model of NAFLD, TRPV2 regulated lipid accumulation in HepG2 cells, and TRPV2 activation inhibited the expression of the cellular senescence markers p21 and p16, all of which were mediated by AMPK phosphorylation. Finally, we found that administration of probenecid, a TRPV2 agonist, impaired HFD-induced hepatic steatosis and suppressed HFD-induced elevation in p21 and p16. Collectively, our findings imply that hepatic TRPV2 protects against the accumulation of lipids by modulating p21 signalling.

Liver fibrosis, as an excess deposition of extracellular matrix (ECM) components, results from chronic liver injury as well as persistent activation of inflammatory response and of fibrogenesis. Liver fibrosis is a major determinant for chronic liver disease (CLD) progression and in the last two decades our understanding on the major molecular and cellular mechanisms underlying the fibrogenic progression of CLD has dramatically improved, boosting pre-clinical studies and clinical trials designed to find novel therapeutic approaches. From these studies several critical concepts have emerged, starting to reveal the complexity of the pro-fibrotic microenvironment which involves very complex, dynamic and interrelated interactions between different hepatic and extrahepatic cell populations. This review will offer first a recapitulation of established and novel pathophysiological basic principles and concepts by intentionally focus the attention on NAFLD/NASH, a metabolic-related form of CLD with a high impact on the general population and emerging as a leading cause of CLD worldwide. NAFLD/NASH-related pro-inflammatory and profibrogenic mechanisms will be analysed as well as novel information on cells, mediators and signalling pathways which have taken advantage from novel methodological approaches and techniques (single cell genomics, imaging mass cytometry, novel in vitro two- and three-dimensional models, etc.). We will next offer an overview on recent advancement in diagnostic and prognostic tools, including serum biomarkers and polygenic scores, to support the analysis of liver biopsies. Finally, this review will provide an analysis of current and emerging therapies for the treatment of NAFLD/NASH patients.

Postprandial hyperglycemia is an early indicator of impaired glucose tolerance that leads to type 2 diabetes mellitus (T2DM). Alterations in the fatty acid composition of phospholipids have been implicated in diseases such as T2DM and nonalcoholic fatty liver disease. Lysophospholipid acyltransferase 10 (LPLAT10, also called LPCAT4 and LPEAT2) plays a role in remodeling fatty acyl chains of phospholipids; however, its relationship with metabolic diseases has not been fully elucidated. LPLAT10 expression is low in the liver, the main organ that regulates metabolism, under normal conditions. Here, we investigated whether overexpression of LPLAT10 in the liver leads to improved glucose metabolism. For overexpression, we generated an LPLAT10-expressing adenovirus (Ad) vector (Ad-LPLAT10) using an improved Ad vector. Postprandial hyperglycemia was suppressed by the induction of glucose-stimulated insulin secretion in Ad-LPLAT10-treated mice compared with that in control Ad vector-treated mice. Hepatic and serum levels of phosphatidylcholine 40:7, containing C18:1 and C22:6, were increased in Ad-LPLAT10-treated mice. Serum from Ad-LPLAT10-treated mice showed increased glucose-stimulated insulin secretion in mouse insulinoma MIN6 cells. These results indicate that changes in hepatic phosphatidylcholine species due to liver-specific LPLAT10 overexpression affect the pancreas and increase glucose-stimulated insulin secretion. Our findings highlight LPLAT10 as a potential novel therapeutic target for T2DM.

Equine metabolic syndrome (EMS) is a significant global health concern in veterinary medicine. There is increasing interest in utilizing molecular agents to modulate hepatocyte function for potential clinical applications. Recent studies have shown promising results in inhibiting protein tyrosine phosphatase (PTP1B) to maintain cell function in various models. In this study, we investigated the effects of the inhibitor Trodusquemine (MSI-1436) on equine hepatic progenitor cells (HPCs) under lipotoxic conditions. We examined proliferative activity, glucose uptake, and mitochondrial morphogenesis. Our study found that MSI-1436 promotes HPC entry into the cell cycle and protects them from palmitate-induced apoptosis by regulating mitochondrial dynamics and biogenesis. MSI-1436 also increases glucose uptake and protects HPCs from palmitate-induced stress by reorganizing the cells' morphological architecture. Furthermore, our findings suggest that MSI-1436 enhances 2-NBDG uptake by increasing the expression of SIRT1, which is associated with liver insulin sensitivity. It also promotes mitochondrial dynamics by modulating mitochondria quantity and morphotype as well as increasing the expression of PINK1, MFN1, and MFN2. Our study provides evidence that MSI-1436 has a positive impact on equine hepatic progenitor cells, indicating its potential therapeutic value in treating EMS and insulin dysregulation.

As the most prevalent chronic liver disease globally, NAFLD encompasses a pathological process that ranges from simple steatosis to NASH, fibrosis, cirrhosis, and HCC, closely associated with numerous extrahepatic diseases. While the initial etiology was believed to be hepatocyte injury caused by lipid toxicity from accumulated triglycerides, recent studies suggest that an imbalance of cholesterol homeostasis is of greater significance. The role of nuclear receptors in regulating liver cholesterol homeostasis has been demonstrated to be crucial. This review summarizes the roles and regulatory mechanisms of nuclear receptors in the 3 main aspects of cholesterol production, excretion, and storage in the liver, as well as their cross talk in reverse cholesterol transport. It is hoped that this review will offer new insights and theoretical foundations for the study of the pathogenesis and progression of NAFLD and provide new research directions for extrahepatic diseases associated with NAFLD.

Qingluotongbi formula (QLT) is a Chinese medicine compound consisting of Tripterygium wilfordii Hook. f. (Celastraceae, TW), Panax notoginseng (Burkill) F.H.Chen (Araliaceae, PN), Rehmannia glutinosa (Gaertn.) DC. (Orobanchaceae, RG), Sinomenium acutum (Thunb.) Rehder & E.H. Wilson (Menispermaceae, SA), and Bombyx mori L. (Bombycidae, BM).

This study investigated the protective effect and possible mechanism of QLT against TW-induced liver injury in mice.

To establish the model of TW-induced liver injury in mice, C57BL/6J mice were randomly divided into 4 groups: control group, low-dose TW group, middle-dose TW group, and high-dose TW group. To observe the effects of QLT and its individual ingredients against TW-induced liver injury, C57BL/6J mice were randomly divided into 7 groups: control group, TW group, QLT group, PN group, RG group, SA group, BM group.After administration for 7 days, C57BL/6J mice were tested for biochemical indicators and liver pathological changes. Then, we evaluated the mitochondrial function and analysed the gene and protein expression related to the peroxisome proliferator-activated receptor alpha (PPARα)/peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) pathway by quantitative real-time PCR (qRT-PCR) and Western blotting.

Compared with the control group (0.30 ± 0.35), TW significantly increased mice liver histological score (L, 0.95 ± 1.14; M, 1.25 ± 1.16; H, 4.00 ± 1.13). QLT and its ingredients significantly improved the pathology scores (CON, 0.63 ± 0.74; TW, 4.19 ± 1.53; QLT, 1.56 ± 0.62; PN, 1.94 ± 0.68; RG, 2.75 ± 1.39; SA, 4.13 ± 0.99; BM, 4.13 ± 0.99). Western blot and qRT-PCR analysis revealed that QLT and its ingredients reversed TW-induced suppression of PPARα/PGC1-α pathway.Discussion and conclusions: These findings provide valuable information for compound compatibility studies and TW clinical applications.

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is the most prevalent liver disease globally, yet no therapies are approved. The effects of Escherichia coli Nissle 1917 expressing aldafermin, an engineered analog of the intestinal hormone FGF19, in combination with dietary change were investigated as a potential treatment for MASLD.

MASLD was induced in C57BL/6J male mice by American lifestyle-induced obesity syndrome diet and then switched to a standard chow diet for seven weeks. In addition to the dietary change, the intervention group received genetically engineered E. coli Nissle expressing aldafermin, while control groups received either E. coli Nissle vehicle or no treatment. MASLD-related plasma biomarkers were measured using an automated clinical chemistry analyzer. The liver steatosis was assessed by histology and bioimaging analysis using Fiji (ImageJ) software. The effects of the intervention in the liver were also evaluated by RNA sequencing and liquid-chromatography-based non-targeted metabolomics analysis. Pathway enrichment studies were conducted by integrating the differentially expressed genes from the transcriptomics findings with the metabolites from the metabolomics results using Ingenuity pathway analysis.

After the intervention, E. coli Nissle expressing aldafermin along with dietary changes reduced body weight, liver steatosis, plasma aspartate aminotransferase, and plasma cholesterol levels compared to the two control groups. The integration of transcriptomics with non-targeted metabolomics analysis revealed the downregulation of amino acid metabolism and related receptor signaling pathways potentially implicated in the reduction of hepatic steatosis and insulin resistance. Moreover, the downregulation of pathways linked to lipid metabolism and changes in amino acid-related pathways suggested an overall reduction of oxidative stress in the liver.

These data support the potential for using engineered microbial therapeutics in combination with dietary changes for managing MASLD.

Nonalcoholic fatty liver disease (NAFLD) and its inflammatory and often progressive subtype nonalcoholic steatohepatitis (NASH), have emerged as significant contributors to hepatic morbidity worldwide. The pathophysiology of NAFLD/NASH is multifaceted, variable, and remains incompletely understood. The pivotal role of liver-resident and recruited macrophages in the pathogenesis of NAFLD and NASH is widely acknowledged as a crucial factor in innate immunity. The remarkable plasticity of macrophages enables them to assume diverse activation and polarization states, dictated by their immunometabolism microenvironment and functional requirements. Recent studies in the field of immunometabolism have elucidated that alterations in the metabolic profile of macrophages can profoundly influence their activation state and functionality, thereby influencing various pathological processes. This review primarily focuses on elucidating the polarization and activation states of macrophages, highlighting the correlation between their metabolic characteristics and the transition from pro-inflammatory to anti-inflammatory phenotypes. Additionally, we explore the potential of targeting macrophage metabolism as a promising therapeutic approach for the management of NAFLD/NASH.

The burden of chronic liver disease is rising substantially worldwide. Fibrosis, characterized by excessive deposition of extracellular matrix proteins, is the common pathway leading to cirrhosis, and limited treatment options are available. There is increasing evidence suggesting the role of cellular stress responses contributing to fibrogenesis. This Review provides an overview of studies that analyse the role of cellular stress in different cell types involved in fibrogenesis, including hepatocytes, hepatic stellate cells, liver sinusoidal endothelial cells and macrophages.

Nonalcoholic fatty liver disease (NAFLD) consists of a broad spectrum of conditions, and nonalcoholic steatohepatitis (NASH) is the advanced form of NAFLD. TAF15 is a DNA and RNA binding protein and is involved in crucial inflammatory signalling pathways. We aimed to investigate the role of TAF15 in the progression of NASH and the underlying molecular mechanism.

We generated mice with hepatocyte-specific knockdown and overexpression of TAF15 using a specific adeno-associated virus (AAV). NASH models were established by feeding mice high-fat and high-cholesterol diets and methionine- and choline-deficient diets. Cleavage under targets and tagmentation and dual-luciferase reporter assays were performed to investigate the effect of TAF15 on FASN transcription. Coimmunoprecipitation and immunofluorescence assays were conducted to explore the interaction of TAF15 and p65. In vitro coculture systems were established to study the interactions of hepatocytes, macrophages and HSCs.

TAF15 was significantly increased in the livers of mouse NASH models and primary hepatocyte NASH model. Knockdown of TAF15 inhibited steatosis, inflammation and fibrosis, while overexpression of TAF15 promoted NASH phenotypes. Mechanistically, TAF15 bound directly to the promoter region of FASN to facilitate its expression, thereby promoting steatosis. Moreover, TAF15 interacted with p65 and activated the NF-κB signalling pathway, increasing the secretion of proinflammatory cytokines and triggering M1 macrophage polarization. Treatment with the FASN inhibitor orlistat partially reversed the phenotypes.

These results suggested that TAF15 exacerbated NASH progression by regulating lipid metabolism and inflammation via transcriptional activation of FASN and interacting with p65 to activate the NF-κB signalling pathway.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease that is becoming increasingly prevalent, and it ranges from simple steatosis to cirrhosis. However, there is still a lack of pharmacotherapeutic strategies approved by the Food and Drug Administration, which results in a higher risk of death related to carcinoma and cardiovascular complications. Of note, it is well established that the pathogenesis of NAFLD is tightly associated with whole metabolic dysfunction. Thus, targeting interconnected metabolic conditions could present promising benefits to NAFLD, according to a number of clinical studies. Here, we summarize the metabolic characteristics of the development of NAFLD, including glucose metabolism, lipid metabolism and intestinal metabolism, and provide insight into pharmacological targets. In addition, we present updates on the progresses in the development of pharmacotherapeutic strategies based on metabolic intervention globally, which could lead to new opportunities for NAFLD drug development.

Obesity is a global epidemic and overwhelming evidence indicates that it is a risk factor for numerous cancers, including hepatocellular carcinoma (HCC), the third leading cause of cancer-related deaths worldwide. Obesity-associated hepatic tumorigenesis develops from nonalcoholic fatty liver disease (NAFLD), progressing to nonalcoholic steatohepatitis (NASH), cirrhosis and ultimately to HCC. The rising incidence of obesity is resulting in an increased prevalence of NAFLD and NASH, and subsequently HCC. Obesity represents an increasingly important underlying etiology of HCC, in particular as the other leading causes of HCC such as hepatitis infection, are declining due to effective treatments and vaccines. In this review, we provide a comprehensive overview of the molecular mechanisms and cellular signaling pathways involved in the pathogenesis of obesity-associated HCC. We summarize the preclinical experimental animal models available to study the features of NAFLD/NASH/HCC, and the non-invasive methods to diagnose NAFLD, NASH and early-stage HCC. Finally, since HCC is an aggressive tumor with a 5-year survival of less than 20%, we will also discuss novel therapeutic targets for obesity-associated HCC and ongoing clinical trials.