Lipid Metabolism In Hepatocytes Research Articles

P-790 Engineering Vascularized Human Endometrial Organoids for In Vivo Tissue Regeneration and Repair Applications

Abstract Study question Is it possible to construct a vascularized endometrial organoid in vitro using endometrial epithelial organoids, endometrial stromal cells, and HUVECs? And its application value? Summary answer We have successfully engineered a vascularized endometrial organoid and demonstrated its remarkable ability to repair damaged endometrium. ScRNA-sequencing revealed intricate regulatory relationships among the cells. What is known already In vivo, endometrial epithelial organoids (EEOs) have been proven effective in repairing damaged endometrium. In vitro, when co-cultured with endometrial stromal cells (ESCs) in matrigel, EEOs self-assemble into endometrial assembloids that accurately replicate the native tissue’s characteristics. Endothelial cells play a critical regulatory role in various physiological processes across different tissues, such as lipid metabolism in hepatocytes, neurogenesis within the brain, and the menstrual cycle regulation in uterine endometrium. By leveraging organ-on-a-chip technology using HUVECs (commonly utilized endothelial cells), it is possible to construct functional and complex organoids with vascularization, including liver buds, brain organoids, and pancreatic islets. Study design, size, duration Different ratios of Fibrin and Matrigel were explored to optimize the hydrogel conditions. The composite medium was optimized with ExM and ECM. Cellular interactions were identified through cell-specific staining. Addition of reproductive hormones was performed to determine if the complexes exhibited hormone-responsive behavior. Transplantation of the organoid complexes into mice with endometrial injury was conducted to assess their repair capacity. Single-cell RNA sequencing was employed to identify cell clusters and analyze intercellular interactions. Participants/materials, setting, methods EEOs, ESCs, and HUVECs self-assemble into HEO complexes. EEOs and ESCs self-assemble into EO complexes as controls. Immunofluorescence and immunohistochemical analysis are conducted to detect cell-specific markers. PBS, Matrigel, EO and HEO complexes are transplanted into mice with endometrial injury. JC-1 staining and transmission electron microscopy are used to verify mitochondrial functionality. Electrical resistance measured by EVOM2, HUVEC tubule formation and sprouting assays are performed to investigate the formation of vascular networks within the complexes. Main results and the role of chance Under dynamic flow conditions of ExM and ECM at a ratio of 5:5, HEO complexes and EO complexes were self-assembled in a fibrin-matrigel hydrogel. The HEO complexes exhibited a capillary network-like structure, intricate cellular interactions, and a transcriptome profile similar to that of the in vivo endometrium. Moreover, HEO complexes responded to sex hormones, facilitating endometrial regeneration and improving the reproductive capacity of mice with endometrial injury. The subcutaneous transplantation of HEO complexes in mice revealed the perfusion of mouse blood within the human blood vessel network in the grafts, highlighting the participation of HUVECs in the vascular formation of the transplanted grafts. ScRNA sequencing analysis of HEO complexes demonstrated a notable rise in the proportion of proliferative subclusters of endometrial epithelial cells and stromal cells compared to EO complexes, especially under estrogen culture conditions. Additionally, HUVECs enhanced the mitochondrial functionality of endometrial cells and remodeled the extracellular matrix. Cell communication analysis revealed that endometrial epithelial and stromal cells enhance the formation of vascular networks in HUVECs within the HEO complexes through WNT7A and WNT5A signaling pathways. Limitations, reasons for caution Due to the time constraints of the study, we did not observe the birth rate of offspring mice following endometrial repair. ScRNA sequencing also uncovered that HUVECs promote ribosomal functionality in endometrial cells within HEO complexes, but further validation of this finding is necessary. Wider implications of the findings The implantation of HEO complexes derived from woman EEOs, ESCs, and HUVECs into the endometrium holds potential for “endometrial re-engineering” in the therapeutic management of Asherman’s syndrome or thin endometrium. Trial registration number not applicable

PFHpA alters lipid metabolism and increases the risk of metabolic dysfunction-associated steatotic liver disease in youth-a translational research framework.

To address the growing epidemic of liver disease, particularly in pediatric populations, it is crucial to identify modifiable risk factors for the development and progression of metabolic dysfunction-associated steatotic liver disease (MASLD). Per- and polyfluoroalkyl substances (PFAS) are persistent ubiquitous chemicals and have emerged as potential risk factors for liver damage. However, their impact on the etiology and severity of MASLD remains largely unexplored in humans. This study aims to bridge the gap between human and in vitro studies to understand how exposure to perfluoroheptanoic acid (PFHpA), one of the emerging PFAS replacements which accumulates in high concentrations in the liver, contributes to MASLD risk and progression. First, we showed that PFHpA plasma concentrations were significantly associated with increased risk of MASLD in obese adolescents. Further, we examined the impact of PFHpA on hepatic metabolism using 3D human liver spheroids and single-cell transcriptomics to identify major hepatic pathways affected by PFHpA. Next, we integrated the in vivo and in vitro multi-omics datasets with a novel statistical approach which identified signatures of proteins and metabolites associated with MASLD development triggered by PFHpA exposure. In addition to characterizing the contribution of PFHpA to MASLD progression, our study provides a novel strategy to identify individuals at high risk of PFHpA-induced MASLD and develop early intervention strategies. Notably, our analysis revealed that the proteomic signature exhibited a stronger correlation between both PFHpA exposure and MASLD risk compared to the metabolomic signature. While establishing a clear connection between PFHpA exposure and MASLD progression in humans, our study delved into the molecular mechanisms through which PFHpA disrupts liver metabolism. Our in vitro findings revealed that PFHpA primarily impacts lipid metabolism, leading to a notable increase of lipid accumulation in human hepatocytes after PFHpA exposure. Among the pathways involved in lipid metabolism in hepatocytes, regulation of lipid metabolism by PPAR-a showed a remarkable activation. Moreover, the translational research framework we developed by integrating human and in vitro data provided us biomarkers to identify individuals at a high risk of MASLD due to PFHpA exposure. Our framework can inform policies on PFAS-induced liver disease and identify potential targets for prevention and treatment strategies.

Effects of Deoxynivalenol and Its Acetylated Derivatives on Lipid Metabolism in Human Normal Hepatocytes

Deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) belong to type B trichothecenes that are widely detected in agricultural products as one of the most common classes of mycotoxins. In the present study, we aimed to characterize the alteration of lipid metabolism in normal human hepatocytes by poisoning with DON and its acetylated derivatives. After verifying the hepatotoxicity of the three toxins, DON, 15-ADON, and 3-ADON, the mRNA expression was determined by transcriptomics, and the results showed that DON and 15-ADON had a significant regulatory effect on the transcriptome, in which glycerophospholipid metabolism pathway and phospholipase D signaling pathways have not been reported in studies of DON and its acetylated derivatives. For further validation, we explored lipid metabolism in depth and found that PC (15:0/16:0), PC (16:1/18:3), PC (18:1/22:6), PC (16:0/16:0), PC (16:0/16:1), PC (16:1/18:1), PC (14:0/18:2), PE (14:0/16:0) and PE (18:1/18:3) were downregulated for all nine lipids. Combined with the transcriptome results, we found that hepatic steatosis induced by the three toxins, DON, 15-ADON and 3-ADON, was associated with altered expression of genes related to lipid oxidation, lipogenesis and lipolysis, and their effects on lipid metabolism in L-02 cells were mainly realized through the PC-PE cycle.

Resveratrol and (-)-Epigallocatechin-3-gallate Regulate Lipid Metabolism by Activating the AMPK Pathway in Hepatocytes.

The purpose of this study was to explore the effects of Res and EGCG on cell growth, cellular antioxidant levels, and cellular lipid metabolism in hepatocytes. In this experiment, leghorn male hepatoma (LMH) cells were used as hepatocytes. The results showed that 6.25-25 μM Res and EGCG had no adverse effects on cell viability and growth. Meanwhile, with the increasing dosage of Res and EGCG, the contents of total cholesterol (TC), total glyceride (TG), and malondialdehyde (MDA) in hepatocytes decreased significantly (p < 0.05), while the contents of glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), and catalase (CAT) increased significantly (p < 0.05). In addition, western blot results showed that Res and EGCG could significantly increase the expression of p-AMPK protein and reduce the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) protein in hepatocytes (p < 0.05). Moreover, q-PCR results showed that with the increase in Res and EGCG, the expression of cholesterol- and fatty acid synthesis-related genes decreased significantly (p < 0.05). In conclusion, Res and EGCG can increase the antioxidant capacity of hepatocytes and reduce the synthesis of TC and TG in hepatocytes by activating AMPK, thereby regulating lipid metabolism in hepatocytes.

Temporal multiscale modeling of biochemical regulatory networks: Calcium-regulated hepatocyte lipid and glucose metabolism

Hepatocyte lipid and glucose metabolism is regulated not only by major hormones like insulin and glucagon but also by many other factors, including calcium ions. Recently, mitochondria-associated membrane (MAM) dysfunction combined with incorrect IP3-receptor regulation has been shown to result in abnormal calcium signaling in hepatocytes. This dysfunction could further lead to hepatic metabolism pathology. However, the exact contribution of MAM dysfunction, incorrect IP3-receptor regulation and insulin resistance to the calcium-insulin-glucagon interplay is not understood yet. In this work, we analyze the role of abnormal calcium signaling and insulin dysfunction in hepatocytes by proposing a model of hepatocyte metabolic regulatory network with a detailed focus on the model construction details besides the biological aspect. In this work, we analyze the role of abnormal calcium signaling and insulin dysfunction in hepatocytes by proposing a model of hepatocyte metabolic regulatory network. We focus on the model construction details, model validation, and predictions. We describe the dynamic regulation of signaling processes by sigmoid Hill function. In particular, we study the effect of both the Hill function slope and the distance between Hill function extremes on metabolic processes in hepatocytes as a model of nonspecific insulin dysfunction. We also address the significant time difference between characteristic time of glucose hepatic processing and a typical calcium oscillation period in hepatocytes. Our modeling results show that calcium signaling dysfunction results in an abnormal increase in postprandial glucose levels, an abnormal glucose decrease in fasting, and a decreased amount of stored glycogen. An insulin dysfunction of glucose phosphorylation, glucose dephosphorylation, and glycogen breakdown also cause a noticeable effect. We also get some insight into the so-called hepatic insulin resistance paradox, confirming the hypothesis regarding indirect insulin action on hepatocytes via dysfunctional adipocyte lipolysis.

Ablation of histone methyltransferase Suv39h2 in hepatocytes attenuates NASH in mice

AimsNon-alcoholic steatohepatitis (NASH) is characterized by aberrant lipid metabolism in hepatocytes. We investigated the involvement of a histone H3K9 methyltransferase Suv39h2 in the pathogenesis of NASH. Methods and materialsNASH is induced by feeding the mice with a high-fat high-carbohydrate (HFHC) diet or a high-fat choline-deficient amino acid defined (HFD-CDAA) diet. The Suv39h2f/f mice were crossbred with the Alb-Cre mice to specifically delete Suv39h2 in hepatocytes. Key findingsAblation of Suv39h2 in hepatocytes improved insulin sensitivity of the mice fed either the HFHC diet or the CDAA-HFD diet. Importantly, Suv39h2 deletion significantly ameliorated NAFLD as evidenced by reduced lipid accumulation, inflammation, and fibrosis in the liver. RNA-seq uncovered Vanin-1 (Vnn1) as a novel transcriptional target for Suv39h2. Mechanistically, Suv39h2 repressed Vnn1 transcription in hepatocytes exposed to free fatty acids. Consistently, Vanin-1 knockdown normalized lipid accumulation in Suv39h2-null hepatocytes. Importantly, a significant correlation between Suv39h2, Vanin-1, and hepatic triglyceride levels was identified in NASH patients. SignificanceOur study uncovers a novel mechanism whereby Suv39h2 may contribute to NASH pathogenesis and suggests that targeting the Suv39h2-Vanin-1 axis may yield novel therapeutic solutions against NASH.

Elevated expression of LCN13 through FXR activation ameliorates hepatocellular lipid accumulation and inflammation

BackgroundLipocalin 13 (LCN13) is a member of the lipocalin family that consists of numerous secretory proteins. LCN13 high-expression has been reported to possess anti-obesity and anti-diabetic effects. Although metabolic dysfunction-associated steatotic liver diseases (MASLD) including metabolic dysfunction-associated steatohepatitis (MASH) are frequently associated with obesity and insulin resistance, the functional role of endogenous LCN13 and the therapeutic effect of LCN13 in MASH and related metabolic deterioration have not been evaluated. MethodsWe employed a methionine-choline deficient diet model and MASH cell models to investigate the role of LCN13 in MASH development. We sought to explore the effects of LCN13 on lipid metabolism and inflammation in hepatocytes under PA/OA exposure using Western blotting, real-time RT-PCR, enzyme-linked immunosorbent assay, hematoxylin and eosin staining, oil red O staining. Using RNA sequencing, chromatin immunoprecipitation assay, and luciferase reporter assays to elucidate whether farnesoid X receptor (FXR) regulates human LCN13 transcription as a transcription factor. ResultsOur study found that LCN13 was down-regulated in MASH patients, MASH mouse and cell models. LCN13 overexpression in hepatocyte cells significantly inhibited lipid accumulation and inflammation in vitro. Conversely, LCN13 downregulation significantly exacerbated lipid accumulation and inflammatory responses in vivo and in vitro. Mechanistically, we provided the first evidence that LCN13 was transcriptionally activated by FXR, representing a novel direct target gene of FXR. And the key promoter region of LCN13 binds to FXR was also elucidated. We further revealed that LCN13 overexpression via FXR activation ameliorates hepatocellular lipid accumulation and inflammation in vivo and in vitro. Furthermore, LCN13-down-regulated mice exhibited aggravated MASH phenotypes, including increased hepatic lipid accumulation and inflammation. ConclusionOur findings provide new insight regarding the protective role of LCN13 in MASH development and suggest an innovative therapeutic strategy for treating MASH or related metabolic disorders.

Differential Disruption of Glucose and Lipid Metabolism Induced by Phthalates in Human Hepatocytes and White Adipocytes.

Phthalic acid esters (PAEs), commonly used as plasticizers, are pervasive in the environment, leading to widespread human exposure. The association between phthalate exposure and metabolic disorders has been increasingly recognized, yet the precise biological mechanisms are not well-defined. In this study, we explored the effects of monoethylhexyl phthalate (MEHP) and monocyclohexyl phthalate (MCHP) on glucose and lipid metabolism in human hepatocytes and adipocytes. In hepatocytes, MEHP and MCHP were observed to enhance lipid uptake and accumulation in a dose-responsive manner, along with upregulating genes involved in lipid biosynthesis. Transcriptomic analysis indicated a broader impact of MEHP on hepatic gene expression relative to MCHP, but MCHP particularly promoted the expression of the gluconeogenesis key enzymes G6PC and FBP1. In adipocytes, MEHP and MCHP both increased lipid droplet formation, mimicking the effects of the Peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone (Rosi). Transcriptomic analysis revealed that MEHP predominantly altered fatty acid metabolism pathways in mature adipocytes (MA), whereas MCHP exhibited less impact. Metabolic perturbations from MEHP and MCHP demonstrate shared activation of the PPARs pathway in hepatocytes and adipocytes, but the cell-type discrepancy might be attributed to the differential expression of PPARγ. Our results indicate that MEHP and MCHP disrupt glucose and lipid homeostasis in human liver and adipose through mechanisms that involve the PPAR and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways, highlighting the nuanced cellular responses to these environmental contaminants.

Effect of high NEFA concentration on lipid metabolism disorders in hepatocytes based on lipidomics.

Introduction: High concentrations of nonesterified fatty acids (NEFA) is the key of characteristic of fatty liver in dairy cows. Therefore, the aim of this study was to investigate the effect of high concentration of NEFA on lipid metabolism in hepatocytes through the lipidomic approach and molecular biology techniques. Methods: Stimulate AML-12 cells with different concentrations of NEFA, observe the cellular lipid accumulation, and select 0.6mM NEFA stimulation concentration for subsequent experiments. Collect cells for lipidomics analysis. Results: High concentration of NEFA (0.6-2.4mM) significantly reduced the cell viability in a concentration-dependent manner, indicating that high concentrations of NEFA have lipotoxicity on hepatocytes. In addition, NEFA promoted triglycerides (TAG) accumulation, increased the mRNA expression of the lipogenic molecules SREBP1c and FASN, and decreased the mRNA expression of lipolytic molecules CPT1A and HSL in hepatocytes. Mechanistically, high concentration of NEFA induced lipid metabolism disorders in hepatocytes by regulating metabolic pathways such as glycerol phospholipid metabolism, glycosyl phosphatidylinositol anchored biosynthesis, triglyceride metabolism, sphingolipid metabolism, and inositol phosphate metabolism. Discussion: High concentration of NEFA is lipotoxic to cells, promoting lipid accumulation. LPE (18:2), LPE (18:3), LPE (18:1) via glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, glycerolipid metabolism, sphingolipid metabolism, and inositol phosphate metabolism, indicating their potential regulation role in the pathogenesis of fatty liver.

Gypenoside XIII regulates lipid metabolism in HepG2 hepatocytes and ameliorates nonalcoholic steatohepatitis in mice.

Gypenoside XIII is isolated from Gynostemma pentaphyllum (Thunb.) Makino. In mice, G. pentaphyllum extract and gypenoside LXXV have been shown to improve non-alcoholic steatohepatitis (NASH). This study investigated whether gypenoside XIII can regulate lipid accumulation in fatty liver cells or attenuate NASH in mice. We used HepG2 hepatocytes to establish a fatty liver cell model using 0.5 mM oleic acid. Fatty liver cells were treated with different concentrations of gypenoside XIII to evaluate the molecular mechanisms of lipid metabolism. In addition, a methionine/choline-deficient diet induced NASH in C57BL/6 mice, which were given 10 mg/kg gypenoside XIII by intraperitoneal injection. In fatty liver cells, gypenoside XIII effectively suppressed lipid accumulation and lipid peroxidation. Furthermore, gypenoside XIII significantly increased SIRT1 and AMPK phosphorylation to decrease acetyl-CoA carboxylase phosphorylation, reducing fatty acid synthesis activity. Gypenoside XIII also decreased lipogenesis by suppressing sterol regulatory element-binding protein 1c and fatty acid synthase production. Gypenoside XIII also increased lipolysis and fatty acid β-oxidation by promoting adipose triglyceride lipase and carnitine palmitoyltransferase 1, respectively. In an animal model of NASH, gypenoside XIII effectively decreased the lipid vacuole size and number and reduced liver fibrosis and inflammation. These findings suggest that gypenoside XIII can regulate lipid metabolism in fatty liver cells and improve liver fibrosis in NASH mice. Therefore, gypenoside XIII has potential as a novel agent for the treatment of NASH.

Vitamin D inhibits tamoxifen-induced non-alcoholic fatty liver disease through a nonclassical estrogen receptor/liver X receptor pathway

Non-alcoholic Fatty Liver Disease (NAFLD) is one of the common side effects of tamoxifen treatment for estrogen receptor-positive breast cancer, and is representative of disorders of energy metabolism. Fatty liver is induced after tamoxifen (TAM) inhibition of estrogen receptor activity, but the exact mechanism is not clear. This study investigated the effects and mechanisms of TAM-induced steatosis in the liver. The effects and mechanisms of TAM on hepatocyte lipid metabolism were assessed using C57BL/6 female mice and human hepatoma cells. TAM promoted fat accumulation in the liver by upregulation of Srebp-1c expression. Regarding the molecular mechanism, TAM promoted the recruitment of the auxiliary transcriptional activator, p300, and dissociated the auxiliary transcriptional repressor, nuclear receptor corepressor (NCOR), of the complexes, which led to enhancement of Srebp-1c transcription and an increase of triglyceride (TG) synthesis. Vitamin D (VD), a common fat-soluble vitamin, can decrease TAM-induced NAFLD by promoting p300 dissociation and NCOR recruitment. Tamoxifen promoted the recruitment and dissociation of co-transcription factors on the LXR/ER/RXR receptor complex, leading to a disorder of liver lipid metabolism. VD interfered with TAM-induced liver lipid metabolism disorders by reversing this process.

Bisphenol A (BPA) exposure aggravates hepatic oxidative stress and inflammatory response under hypertensive milieu – Impact of low dose on hepatocytes and influence of MAPK and ER stress pathways

Human exposure to the hazardous chemical, Bisphenol A (BPA), is almost ubiquitous. Due to the prevalence of hypertension (CVD risk factor) in the aged human population, it is necessary to explore its adverse effect in hypertensive subjects. The current study exposed the Nω-nitro-l-arginine methyl ester (L-NAME) induced hypertensive Wistar rats to human exposure relevant low dose of BPA (50 μg/kg) for 30 days period. The liver biochemical parameters, histopathology, immunohistochemistry, gene expression (RT-qPCR), trace elements (ICP-MS), primary rat hepatocytes cell culture and metabolomic (1H NMR) assessments were performed. Results illustrate that BPA exposure potentiates/aggravates hypertension induced tissue abnormalities (hepatic fibrosis), oxidative stress, ACE activity, malfunction of the antioxidant system, lipid abnormalities and inflammatory factor (TNF-α and IL-6) expression. Also, in cells, BPA increased ROS generation, mitochondrial dysfunction and lipid peroxidation without any impact on cytotoxicity and caspase 3 and 9 activation. Notably, BPA exposure modulate lipid metabolism (cholesterol and fatty acid) in primary hepatocytes. Finally, the influence of ERK1/2, p38MAPK, ER stress and oxidative stress during relatively high dose of BPA elicited cytotoxicity was observed. Therefore, a precise hazardous risk investigation of BPA exposure in hypertensive populations is highly recommended.

Si-Ni-San inhibits hepatic Fasn expression and lipid accumulation in MAFLD mice through AMPK/p300/SREBP-1c axis

BackgroundSoothing the liver and regulating qi is one of the core ideas of traditional Chinese medicine (TCM) in the treatment of fatty liver. Si-Ni-San (SNS) is a well-known herbal formula in TCM for liver soothing and qi regulation in fatty liver treatment. However, its efficacy lacks modern scientific evidence. PurposeThis study was aimed to investigate the impact of SNS on metabolic associated fatty liver disease (MAFLD) in mice and explore the underlying molecular mechanisms, particularly its effects on lipid metabolism in hepatocytes. MethodsThe therapeutic effect of SNS was evaluated using in vivo and in vitro models of high-fat/high-cholesterol (HFHC) diet-induced mice and palmitic acid (PA)-induced hepatocytes, respectively. Molecular biological techniques such as RNA-sequencing (RNA-seq), co-immunoprecipitation (co-IP), and western blotting were employed to elucidate the molecular mechanism of SNS in regulating lipid metabolism in hepatocytes. ResultsOur findings revealed that SNS effectively reduced lipid accumulation in the livers of HFHC diet-induced mice and PA-induced hepatocytes. RNA-seq analysis demonstrated that SNS significantly down-regulated the expression of fatty acid synthase (Fasn) in the livers of HFHC-fed mice. Mechanistically, SNS inhibited Fasn expression and lipid accumulation by activating adenosine monophosphate (AMP)-activated protein kinase (AMPK). Activation of AMPK suppressed the activity of the transcriptional coactivator p300 and modulated the protein stability of sterol regulatory element-binding protein-1c (SREBP-1c). Importantly, p300 was required for the inhibition of Fasn expression and lipid accumulation by SNS. Furthermore, SNS activated AMPK by decreasing adenosine triphosphate (ATP) production in hepatocytes. ConclusionThis study provided novel evidence on the regulatory mechanisms underlying the effects of SNS on Fasn expression. Our findings demonstrate, for the first time, that SNS exerts suppressive effects on Fasn expression through modulation of the AMPK/p300/SREBP-1c axis. Consequently, this regulatory pathway mitigates excessive lipid accumulation and ameliorates MAFLD in mice.

Inositol-requiring enzyme 1α and c-Jun N-terminal kinase axis activation contributes to intracellular lipid accumulation in calf hepatocytes

During the perinatal period, dairy cows undergo negative energy balance, resulting in elevated circulating levels of nonesterified fatty acids (NEFA). Although increased blood NEFA concentrations are a physiological adaptation of early lactation, excessive NEFA in dairy cows is a major cause of fatty liver. Aberrant lipid metabolism leads to hepatic lipid accumulation and subsequently the development of fatty liver. Both inositol-requiring enzyme 1α (IRE1α) and c-Jun N-terminal kinase (JNK) have been validated for their association with hepatic lipid accumulation, including their regulatory functions in calf hepatocyte insulin resistance, oxidative stress, and apoptosis. Meanwhile, both IRE1α and JNK are involved in lipid metabolism in nonruminants. Therefore, the aim of this study was to investigate how IRE1α and JNK regulate lipid metabolism in bovine hepatocytes. An experiment was conducted on randomly selected 10 healthy cows (hepatic triglyceride [TG] content <1%) and 10 cows with fatty liver (hepatic TG content >5%). Liver tissue and blood samples were collected from experimental cows. Serum concentrations of NEFA and β-hydroxybutyrate (BHB) were greater, whereas serum concentrations of glucose and milk production were lower in cows with fatty liver. The western blot results revealed that dairy cows with fatty liver had higher phosphorylation levels of JNK, c-Jun, and IRE1α in the liver tissue. Three in vitro experiments were conducted using primary calf hepatocytes isolated from 5 healthy calves (body weight: 30-40 kg; 1 d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h, which showed that the phosphorylated levels of JNK, c-Jun, and IRE1α increased in both linear and quadratic effects. In the second experiment, hepatocytes were treated with high concentrations of NEFA (1.2 mM) for 12 h with or without SP600125, a canonical inhibitor of JNK. Western blot results showed that SP600125 treatment could decrease the expression of lipogenesis-associated proteins (PPARγ and SREBP-1c) and increase the expression of fatty acid oxidation (FAO)-associated proteins (CPT1A and PPARα) in NEFA-treated hepatocytes. The perturbed expression of lipogenesis-associated genes (FASN, ACACA, and CD36) and FAO-associated gene ACOX1 were also recovered by JNK inhibition, indicating that JNK reduced excessive NEFA-induced lipogenesis and FAO dysregulation in calf hepatocytes. Third, short hairpin RNA targeting IRE1α (sh-IRE1α) was transfected into calf hepatocytes to silence IRE1α, and KIRA6 was used to inhibit the kinase activity of IRE1α. The blockage of IRE1α could at least partially suppressed NEFA-induced JNK activation. Moreover, the blockage of IRE1α downregulated the expression of lipogenesis genes and upregulated the expression of FAO genes in NEFA-treated hepatocytes. In conclusion, these findings indicate that targeting the IRE1α-JNK axis can reduce NEFA-induced lipid accumulation in bovine hepatocytes by modulating lipogenesis and FAO. This may offer a prospective therapeutic target for fatty liver in dairy cows.

Shenge Formula attenuates high-fat diet-induced obesity and fatty liver via inhibiting ACOX1

BackgroundNonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. Shenge Formula (SGF) is a traditional Chinese medicine that has been used in the clinical treatment of NAFLD, and its therapeutic potential in patients and NAFLD animal models has been demonstrated in numerous studies. However, its underlying mechanism for treating NAFLD remains unclear. PurposeThe aim of this study was to investigate the mechanism of SGF in the treatment of NAFLD using the proteomics strategy. MethodsUltra-high performance liquid chromatography-mass spectrometry (UPLC-MS) was used to determine the main components of SGF. A mouse model of nonalcoholic fatty liver disease was constructed by feeding mice with a high-fat diet for 16 weeks. SGF was administered for an additional 8 weeks, and metformin was used as a positive control. Liver sections were subjected to histopathological assessments. LC-MS/MS was used for the label-free quantitative proteomic analysis of liver tissues. Candidate proteins and pathways were validated both in vivo and in vitro through qRT-PCR, western blot, and immunohistochemistry. The functions of the validated pathways were further investigated using the inhibition strategy. ResultsThirty-nine ingredients were identified in SGF extracts, which were considered to be key compounds in the treatment of NAFLD. SGF administration attenuated obesity and fatty liver by reducing the body weight and liver weight in HFD-fed mice. It also relieved HFD-induced insulin resistance. More importantly, hepatic steatosis was significantly attenuated by SGF administration both in vivo and in vitro. Proteomic profiling of mouse liver tissues identified 184 differential expressed proteins (DEPs) associated with SGF treatment. Bioinformatic analysis of DEPs revealed that regulating the lipid metabolism and energy consumption process of hepatocytes was the main role of SGF in NAFLD treatment. This also indicated that ACOX1 might be the potential target of SGF, which was subsequently verified both in vitro and in vivo. The results demonstrated that SGF inhibited ACOX1 activity, thereby activating PPARα and upregulating CPT1A expression. Increased CPT1A expression promoted mitochondrial β-oxidation, leading to reduced lipid accumulation in hepatocytes. ConclusionsOverall, our findings confirmed the protective effect of SGF against NAFLD and revealed the underlying molecular mechanism of regulating lipid metabolism.

SBC (Sanhuang Xiexin Tang combined with Baihu Tang plus Cangzhu) alleviates NAFLD by enhancing mitochondrial biogenesis and ameliorating inflammation in obese patients and mice

In the context of non-alcoholic fatty liver disease (NAFLD), characterized by dysregulated lipid metabolism in hepatocytes, the quest for safe and effective therapeutics targeting lipid metabolism has gained paramount importance. Sanhuang Xiexin Tang (SXT) and Baihu Tang (BHT) have emerged as prominent candidates for treating metabolic disorders. SXT combined with BHT plus Cangzhu (SBC) has been used clinically for Weihuochisheng obese patients. This retrospective analysis focused on assessing the anti-obesity effects of SBC in Weihuochisheng obese patients. We observed significant reductions in body weight and hepatic lipid content among obese patients following SBC treatment. To gain further insights, we investigated the effects and underlying mechanisms of SBC in HFD-fed mice. The results demonstrated that SBC treatment mitigated body weight gain and hepatic lipid accumulation in HFD-fed mice. Pharmacological network analysis suggested that SBC may affect lipid metabolism, mitochondria, inflammation, and apoptosis—a hypothesis supported by the hepatic transcriptomic analysis in HFD-fed mice treated with SBC. Notably, SBC treatment was associated with enhanced hepatic mitochondrial biogenesis and the inhibition of the c-Jun N-terminal kinase (JNK)/nuclear factor-kappa B (NF-κB) and extracellular signal-regulated kinase (ERK)/NF-κB pathways. In conclusion, SBC treatment alleviates NAFLD in both obese patients and mouse models by improving lipid metabolism, potentially through enhancing mitochondrial biogenesis. These effects, in turn, ameliorate inflammation in hepatocytes.

The Coumarin-Derivative Esculetin Protects against Lipotoxicity in Primary Rat Hepatocytes via Attenuating JNK-Mediated Oxidative Stress and Attenuates Free Fatty Acid-Induced Lipid Accumulation.

Coumarin derivates have been proposed as a potential treatment for metabolic-dysfunction-associated fatty liver disease (MAFLD). However, the mechanisms underlying their beneficial effects remain unclear. In the present study, we explored the potential of the coumarin derivate esculetin in MAFLD, focusing on hepatocyte lipotoxicity and lipid accumulation. Primary cultures of rat hepatocytes were exposed to palmitic acid (PA) and palmitic acid plus oleic acid (OA/PA) as models of lipotoxicity and lipid accumulation, respectively. Esculetin significantly reduced oxidative stress in PA-treated hepatocytes, as shown by decreased total reactive oxygen species (ROS) and mitochondrial superoxide production and elevated expression of antioxidant genes, including Nrf2 and Gpx1. In addition, esculetin protects against PA-induced necrosis. Esculetin also improved lipid metabolism in primary hepatocytes exposed to nonlipotoxic OA/PA by decreasing the expression of the lipogenesis-related gene Srebp1c and increasing the expression of the fatty acid β-oxidation-related gene Ppar-α. Moreover, esculetin attenuated lipid accumulation in OA/PA-treated hepatocytes. The protective effects of esculetin against lipotoxicity and lipid accumulation were shown to be dependent on the inhibition of JNK and the activation of AMPK, respectively. We conclude that esculetin is a promising compound to target lipotoxicity and lipid accumulation in the treatment of MAFLD.

Antihyperlipidemic effect of bread fortified with potato peel powder against Triton X-100-induced hyperlipidemia in male albino rats

The present investigation aimed to improve the nutritional and healthy values and evaluate the influence of bread fortified with dried potato peels (BPP) on Triton X-100-induced hyperlipidemia in male albino rats. The findings demonstrated that BPP lowered hepatic triglycerides and total cholesterol while increasing high-density lipoprotein (HDL-C). BPP exhibited potential lipid-lowering and hepatoprotective actions that were generated by the reduction of oxidative damage and preservation of the non-enzymatic and enzymatic antioxidant systems compromised by hyperlipidemic condition. Furthermore, BPP considerably enhanced the mRNA expression of AMPKα (AMP-activated protein kinase-alpha), suppressed the expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase alpha (ACCα), and decreased the mRNA expression of the adipogenic transcription factor SREBP-1c (sterol regulatory element-binding protein-1c), which are essential regulators of hepatocyte lipid metabolism. A histopathological assessment of the liver confirmed the results. In conclusion, this investigation revealed a positive impact of bread fortified with BPP therapy in preventing hyperlipidemia.

Acute murine cytomegalovirus infection boosts cell-type specific response and lipid metabolism changes in the liver of infant mice.

Human cytomegalovirus (HCMV) infection in infants can lead to severe diseases, including neonatal hepatitis. The single-cell dimensional changes in immune cells after the initial CMV infection remain elusive, as do the effects of CMV infection on hepatic lipid metabolism. We employed single-cell RNA-sequencing to investigate the changes in liver cell types and immune responses in infant mice following murine CMV (MCMV) infection. Additionally, we examined alterations in protein expression profiles related to lipid metabolism in hepatocytes and the role of the key transcription factor PPAR-γ in hepatocytes during CMV infection. Our study revealed that MCMV infects most liver cell types in infant mice, leading to an increase in the proportion of proliferating CD8 effector T cells and a subset of Nos2+ monocytes, potentially playing an essential role in early anti-viral responses. Furthermore, MCMV infection resulted in altered protein expression of lipid metabolism in hepatocytes. Knocking down the transcription factor PPAR-γ in hepatocytes effectively inhibited CMV infection. Our findings underscore the immune system's response to early-stage MCMV infection and the subsequent impact on hepatic lipid metabolism in infant mice. This research provides new insights into the mechanisms of CMV infection and could pave the way for novel therapeutic strategies.

LINC01705 derived from adipocyte exosomes regulates hepatocyte lipid accumulation via an miR-552-3p/LXR axis.

High glucose increases the accumulation of lipid droplets in hepatocytes, which eventually results in nonalcoholic fatty liver disease in patients with diabetes. However, the specific mechanism or communication between adipocyte and hepatocyte lipid metabolism is still ambiguous. In this study, exosomes released from human adipocytes were isolated and identified by their morphology, size, and marker proteins by using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting (WB). Gene expression was detected by qRT-PCR and WB. Lipid accumulation was determined by oil red O staining and analyses of total cholesterol (TC) and triglyceride (TG) content. Our results showed that co-culture of HepG2 cells with adipocytes under high glucose conditions stimulated lipid deposition and LINC01705 expression in the HepG2 cells. Exosomes extracted from adipocytes cultured under high glucose conditions had higher levels of LINC01705 than exosomes extracted from adipocytes cultured under normal glucose conditions. Moreover, LINC01705 expression was also elevated in exosomes extracted from diabetes patients when compared with exosomes isolated from normal volunteers, and exosomes from patients who had diabetes complicated with fatty liver (DCFL) had the highest levels of LINC01705 expression. Treatment of HepG2 cells with exosomes extracted from high glucose-stimulated adipocytes promoted lipid deposition and LINC01705 expression in HepG2 cells. Further experiments showed that overexpression of LINC01705 promoted HepG2 lipid metabolism, while inhibition of LINC01705 had the opposite effect. Mechanistically, LINC01705 competitively bound to miR-552-3p, and treatment with miR-552-3p inhibitor reversed the effects induced by LINC01705 knockdown. Moreover, miR-552-3p was found to regulate the transcription activity of LXRα and thereby modulate lipid metabolism-related gene expression. When taken together, our findings showed that high glucose increased the LINC01705 levels in adipocyte exosomes, and thereby improved HepG2 lipid accumulation via an miR-552-3p/LXR axis.