The mechanism of increased intestinal palmitic acid absorption and its impact on hepatic stellate cell activation in nonalcoholic steatohepatitis.
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ABSTRACT:
SUBMITTER: Hanayama M
PROVIDER: S-EPMC8239050 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Emerging data suggest that changes in intestinal permeability and increased gut microbial translocation contribute to the inflammatory pathway involved in nonalcoholic steatohepatitis (NASH) development. Numerous studies have investigated the association between increased intestinal permeability and NASH. Our meta-analysis of this association investigates the underlying mechanism.A meta-analysis was performed to compare the rates of increased intestinal permeability in patients with NASH and healthy controls. To further address the underlying mechanism of action, we studied changes in intestinal permeability in a diet-induced (methionine-and-choline-deficient; MCD) murine model of NASH. In vitro studies were also performed to investigate the effect of MCD culture medium at the cellular level on hepatocytes, Kupffer cells, and intestinal epithelial cells.Nonalcoholic fatty liver disease (NAFLD) patients, and in particular those with NASH, are more likely to have increased intestinal permeability compared with healthy controls. We correlate this clinical observation with in vivo data showing mice fed an MCD diet develop intestinal permeability changes after an initial phase of liver injury and tumor necrosis factor-? (TNF?) induction. In vitro studies reveal that MCD medium induces hepatic injury and TNF? production yet has no direct effect on intestinal epithelial cells. Although these data suggest a role for hepatic TNF? in altering intestinal permeability, we found that mice genetically resistant to TNF?-myosin light chain kinase (MLCK)-induced intestinal permeability changes fed an MCD diet still develop increased permeability and liver injury.Our clinical and experimental results strengthen the association between intestinal permeability increases and NASH and also suggest that an early phase of hepatic injury and inflammation contributes to altered intestinal permeability in a fashion independent of TNF? and MLCK.
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Project description:Advanced hepatic fibrosis, driven by the activation of hepatic stellate cells (HSCs), affects millions worldwide and is the strongest predictor of mortality in nonalcoholic steatohepatitis (NASH); however, there are no approved antifibrotic therapies. To identify antifibrotic drug targets, we integrated progressive transcriptomic and morphological responses that accompany HSC activation in advanced disease using single-nucleus RNA sequencing and tissue clearing in a robust murine NASH model. In advanced fibrosis, we found that an autocrine HSC signaling circuit emerged that was composed of 68 receptor-ligand interactions conserved between murine and human NASH. These predicted interactions were supported by the parallel appearance of markedly increased direct stellate cell-cell contacts in murine NASH. As proof of principle, pharmacological inhibition of one such autocrine interaction, neurotrophic receptor tyrosine kinase 3-neurotrophin 3, inhibited human HSC activation in culture and reversed advanced murine NASH fibrosis. In summary, we uncovered a repertoire of antifibrotic drug targets underlying advanced fibrosis in vivo. The findings suggest a therapeutic paradigm in which stage-specific therapies could yield enhanced antifibrotic efficacy in patients with advanced hepatic fibrosis.
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Project description:Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline-deficient, high-fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.
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Project description:Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline deficient, high fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.
| S-EPMC10187148 | biostudies-literature
Project description:In the present study, we sought to identify long noncoding RNA (lncRNA) expression profiles in nonalcoholic steatohepatitis (NASH) patients with histologic evidence of lobular inflammation and advanced fibrosis. We profiled lncRNA expression using RNA-sequencing of wedge liver biopsies from 24 nonalcoholic fatty liver disease (NAFLD) patients with normal liver histology, 53 NAFLD patients with lobular inflammation, and 65 NAFLD patients with advanced fibrosis. Transcript profiling identified 4432 and 4057 differentially expressed lncRNAs in comparisons of normal tissue with lobular inflammation and fibrosis samples, respectively. Functional enrichment analysis revealed lncRNA participation in transforming growth factor beta 1 and tumor necrosis factor signaling, insulin resistance, and extracellular matrix maintenance. Several lncRNAs were highly expressed in fibrosis relative to normal tissue, including nuclear paraspeckle assembly transcript 1, hepatocellular carcinoma upregulated lncRNA, and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Two potential target mRNAs, syndecan 4 (SDC4), and C-X-C motif chemokine ligand 5 (CXCL5) were identified for hepatocellular carcinoma upregulated lncRNA and MALAT1, respectively, but only CXCL5 showed differential expression among the different histologic classes. Knockdown of MALAT1 expression reduced CXCL5 transcript and protein levels by 50% and 30%, respectively, in HepG2 cells. The expression of MALAT1 and CXCL5 was upregulated in activated hepatic stellate (LX-2) cells compared to cells in the quiescent state, and MALAT1 expression was regulated by hyperglycemia and insulin in HepG2 cells, but only by insulin in LX-2 cells. Dysregulated lncRNA expression is associated with inflammation and fibrosis in NASH. Functionally relevant differences in MALAT1 expression may contribute to the development of fibrosis in NASH through mechanisms involving inflammatory chemokines.
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Project description:Nonalcoholic fibrosing steatohepatitis is a uniform process throughout nonalcoholic fatty liver disease (NAFLD). MicroRNAs (miRNAs) have been suggested to modulate cellular processes in liver diseases. However, the functional role of miRNAs in nonalcoholic fibrosing steatohepatitis is largely unclear. In this study, we systematically analyzed the hepatic miRNAs by microarray analysis in nonalcoholic fibrosing steatohepatitis in C57BL/6J mice induced by methionine-choline deficient (MCD) diet. We identified 19 up-regulated and 18 down-regulated miRNAs in liver with fibrosis. Among these dysregulated miRNAs, miR-146a-5p was the most significant down-regulated miRNA. Luciferase activity assay confirmed that Wnt1 and Wnt5a were both the target genes of miR-146a-5p. Hepatic miR-146a-5p was down-regulated in fibrosing steatohepatitis, but its target genes Wnt1 and Wnt5a and their consequent effectors α-SMA and Col-1 were significantly up-regulated. In addition, miR-146a-5p was downregulated, whilst Wnt1 and Wnt5a were up-regulated in the activated primary hepatic stellate cells (HSCs) compared to the quiescent primary HSCs. Overexpression of miR-146a-5p in HSCs inhibited HSC activation and proliferation, which concomitant with the decreased expressions of Wnt1, Wnt5a, α-SMA and Col-1. In conclusion, miR-146a-5p suppresses activation and proliferation of HSCs in the progress of nonalcoholic fibrosing steatohepatitis through targeting Wnt1 and Wnt5a and consequent effectors α-SMA and Col-1.
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Project description:Non-alcoholic fatty liver disease (NAFLD) is a dominant cause of chronic liver disease, but the exact mechanism of progression from simple steatosis to nonalcoholic steatohepatitis (NASH) remains unknown. Here, we investigated the role of exosomes in NAFLD progression. Exosomes were isolated from a human hepatoma cell line treated with palmitic acid (PA) and their miRNA profiles examined by microarray. The human hepatic stellate cell (HSC) line (LX-2) was then treated with exosome isolated from hepatocytes. Compared with controls, PA-treated hepatocytes displayed significantly increased CD36 and exosome production. The microarray analysis showed there to be distinctive miRNA expression patterns between exosomes from vehicle- and PA-treated hepatocytes. When LX-2 cells were cultured with exosomes from PA-treated hepatocytes, the expression of genes related to the development of fibrosis were significantly amplified compared to those treated with exosomes from vehicle-treated hepatocytes. In conclusion, PA treatment enhanced the production of exosomes in these hepatocytes and changed their exosomal miRNA profile. Moreover, exosomes derived from PA-treated hepatocytes caused an increase in the expression levels of fibrotic genes in HSCs. Therefore, exosomes may have important roles in the crosstalk between hepatocytes and HSCs in the progression from simple steatosis to NASH.
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Project description:Background & aimsSteatohepatitis drives fibrogenesis in alcohol-related liver disease. Recent studies have suggested that hepatic stellate cells (HSCs) may regulate the parenchymal cell injury and inflammation that precedes liver fibrosis, although the mechanism remains incompletely defined. Neuropilin-1 (NRP-1) and synectin are membrane proteins implicated in HSC activation. In this study, we disrupted NRP-1 and synectin as models to evaluate the role of HSC activation on the development of steatohepatitis in response to alcohol feeding in mice.MethodsMice with HSC-selective deletion of NRP (ColCre/Nrp1loxP) or synectin (ColCre/synectinloxP) vs. paired Nrp1loxP or synectinloxP mice were fed a control diet or the chronic/binge alcohol feeding model. Several markers of steatosis and inflammation were evaluated.ResultsColCre/Nrp1loxP mice showed less fibrosis, as expected, but also less inflammation and steatosis, with lower hepatic triglyceride content. Similar results were observed in the synectin model. Hepatocytes treated with supernatant of HSCs from ColCre/Nrp1loxP mice compared to supernatant from Nrp1loxP mice were protected against ethanol-induced lipid droplet formation. An adipokine and inflammatory protein array from the supernatant of HSCs with NRP-1 knockdown showed a significant reduction in Igfbp3 (a major insulin-like growth factor-binding protein with multiple metabolic functions) and an increase in SerpinA12 (a serine-protease inhibitor) secretion compared to wild-type HSCs. Recombinant Igfbp3 induced lipid droplets, triglyceride accumulation, and lipogenic genes in hepatocytes in vitro, while SerpinA12 was protective against ethanol-induced steatosis. Finally, Igfbp3 was increased, and SerpinA12 was decreased in serum and liver tissue from patients with alcoholic hepatitis.ConclusionSelective deletion of NRP-1 from HSCs attenuates alcohol-induced steatohepatitis through regulation of Igfbp3 and SerpinA12 signaling.Lay summaryHepatic stellate cells are known for their role in fibrosis (scarring of the liver). In this study, we describe their role in the modulation of fat deposition and inflammation in the liver, which occurs secondary to alcohol damage.
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Project description:Lipoprotein lipase (LPL) plays a central role in incorporating plasma lipids into tissues and regulates lipid metabolism and energy balance in the human body. Conversely, LPL expression is almost absent in normal adult livers. Therefore, its physiological role in the liver remains unknown. We aimed to elucidate the role of LPL in the pathophysiology of nonalcoholic steatohepatitis (NASH), a hepatic manifestation of obesity. Hepatic stellate cell (HSC)-specific LPL-knockout (LplHSC-KO ) mice, LPL-floxed (Lplfl/fl ) mice, or double-mutant toll-like receptor 4-deficient (Tlr4-/- ) LplHSC-KO mice were fed a high-fat/high-cholesterol diet for 4 weeks to establish the nonalcoholic fatty liver model or an high-fat/high-cholesterol diet for 24 weeks to establish the NASH model. Human samples, derived from patients with nonalcoholic fatty liver disease, were also examined. In human and mouse NASH livers, serum obesity-related factors, such as free fatty acid, leptin, and interleukin-6, dramatically increased the expression of LPL, specifically in HSCs through signal transducer and activator of transcription 3 signaling, as opposed to that in hepatocytes or hepatic macrophages. In the NASH mouse model, liver fibrosis was significantly reduced in LplHSC-KO mice compared with that in Lplfl/fl mice. Nonenzymatic LPL-mediated cholesterol uptake from serum lipoproteins enhanced the accumulation of free cholesterol in HSCs, which amplified TLR4 signaling, resulting in the activation of HSCs and progression of hepatic fibrosis in NASH. Conclusion: The present study reveals the pathophysiological role of LPL in the liver, and furthermore, clarifies the pathophysiology in which obesity, as a background factor, exacerbates NASH. The LPL-mediated HSC activation pathway could be a promising therapeutic target for treating liver fibrosis in NASH.
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