Transcriptomic Analysis on Lipid Loaded HepaRG Model for Steatosis Reveal the Influence on the Regulation of Drug Metabolizing Enzymes and Transporters
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE122151 | GEO | 2020/12/31
REPOSITORIES: GEO
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Project description:Non-alcoholic fatty liver disease (NAFLD) is a major public health burden and it covers a spectrum of diseases. NAFLD starts with the accumulation of lipid droplets (LDs) within hepatocytes (steatosis). Part of the challenge of studying the mechanistic processes involved in LD accumulation and their implications on the pathogenesis of human NAFLD is due to the available models. Investigating hepatic LDs in humans is challenging and relies on liver biopsies, meaning only cross-sectional data be obtained. On the other hand, LD patterns in in vitro models are poorly defined and rarely reported. Diacylgylcerol acyltransferase (DGAT)2 is one of two enzymes that carry out the final committed step in triacylglycerol (TAG) synthesis. It is unclear whether the enzymes are able to compensate for each other or whether they have distinct roles. It has been hypothesised that DGAT1 primarily utilises exogenous fatty acids and DGAT2 uses de novo-derived fatty acids. Given the important role of this enzyme in TAG synthesis and accumulation, the aims of this study are first to create a cellular model of intrahepatocellular TAG accumulation by manipulating nutritional substrates and to investigate intracellular metabolism in wildtype and DGAT2 knockout cells under these conditions. The experimental workflow for this study is as follows: Huh7 cells (either wild type or knockout) were grown in media containing 11 mM glucose and 2% human serum (HS) for seven days before additional sugars and fatty acids (FAs) were added for a further seven days. All treatments contained 11 mM glucose and 2% HS, either with 200 µM FAs (low fat low sugar; LFLS), 5.5 mM fructose + 200 µM FAs (low fat high sugar; LFHS) or 5.5 mM fructose + 800 µM FAs (high fat high sugar; HFHS). FA metabolism, lipid droplet characteristics and transcriptomic signatures were investigated.
2020-08-21 | E-MTAB-7884 | biostudies-arrayexpress
Project description:ANGPTL4 regulates plasma triglyceride levels by inhibiting lipoprotein lipase. Inactivation of ANGPTL4 decreases plasma triglycerides and reduces risk of coronary artery disease. Unfortunately, targeting ANGPTL4 for the therapeutic management of dyslipidemia and atherosclerosis is hampered by the observation that mice and monkeys in which ANGPTL4 is inactivated exhibit lipid accumulation in mesenteric lymph nodes. In mice these pathological events exclusively unfold upon feeding a high saturated fatty acid diet and are followed by an ultimately lethal pro-inflammatory response and chylous ascites. Here we show that Angptl4-/- mice fed a diet rich in trans fatty acids develop numerous lipid-filled giant cells in their mesenteric lymph nodes, yet do not have elevated serum amyloid and haptoglobin, do not exhibit ascites, and survive, unlike Angptl4-/- mice fed a saturated fatty acid-rich diet. In RAW264.7 macrophages the saturated fatty acid palmitate markedly increases markers of inflammation and the unfolded protein response, whereas the trans-unsaturated elaidate and the cis-unsaturated oleate have the opposite effect. In conclusion, trans and saturated fatty acids have very distinct biological effects. Furthermore, lipid accumulation in mesenteric lymph nodes is uncoupled from activation of an acute-phase response and chylous ascites, suggesting that ANGPTL4 should not be fully dismissed as target for dyslipidemia.
2017-04-27 | GSE98303 | GEO
Project description:Dietary lipids can affect metabolic health through gut microbiota-mediated mechanisms, but the influence of lipid-microbiota interaction on liver steatosis is unknown. We investigated the effect of dietary lipid composition on human microbiota in an observational study and combined diet experiments with microbiota transplants to study lipid-microbiota interactions and liver status in mice. In humans, low intake of saturated fatty acids (SFA) was associated with increased microbial diversity independent of fiber intake. In mice, cecum levels of SFA correlated negatively with microbial diversity and were associated with a shift in butyrate and propionate producers. Mice fed poorly absorbed SFA had improved metabolism and liver status. These features were transmitted by microbial transfer. Diets enriched in n-6- and/or n-3-polyunsaturated fatty acids were protective against steatosis but had minor influence on the microbiota. In summary, we find that unabsorbed SFA correlate with microbiota features that may be targeted to decrease liver steatosis.
2023-08-03 | GSE222060 | GEO
Project description:The oleaginous green microalga Neochloris oleoabundans accumulates both starch and lipids to high levels under stress conditions such as nitrogen starvation. In order to steer the metabolism towards starch or lipids only, it is important to understand the mechanisms behind it. In this study physiological changes and gene expression upon nitrogen starvation were analysed in controlled flat-panel photobioreactors over both short- and long-term time-scales. Starch accumulation is transient and occurs rapidly within 24 hrs upon starvation, while lipid accumulation lasts longer and reaches a maximum after 4 days. The major fraction of accumulated lipids is composed of de novo synthesized neutral lipids - triacylglycerides (TAG) - and is characterized by a decreased composition of the polyunsaturated fatty acids (PUFAs) C18:3 and C16:3 and an increased composition of the mono-unsaturated and saturated fatty acids C18:1/C16:1 and C18:0/C16:0, respectively. RNA-sequencing revealed that genes related to starch biosynthesis and degradation show different temporal expression dynamics compared to those of lipid biosynthesis. An immediate rapid increase in starch synthesis gene expression is followed by an increase in starch degradation and decrease in starch synthesis gene expression. In contrast, increased gene expression for fatty acid and TAG synthesis is initiated later and occurs more gradually. Expression of several fatty acid desaturase (FAD) genes was decreased upon starvation, which corresponds to the observed changes to higher levels of mono-unsaturated and saturated fatty acids. Moreover, several homologs of transcription regulators that were implicated in controlling starch and lipid metabolism in other microalgae showed differential gene expression and might be key regulators of starch and lipid metabolism in N. oleoabundans as well. Promising candidates for future metabolic engineering are a DYRKP homolog that in Chlamydomonas acts as a negative regulator of carbon storage and photosynthetic efficiency under N-starvation, and two bZIP-type regulators that have been implicated to control several steps in microalgal TAG synthesis. Our data for the first time show the temporal dynamics of storage compound accumulation and transcriptional changes on a short- and long-term time-scale during nitrogen starvation in N. oleoabundans, and increases insight into the genetic foundation for starch and lipid metabolism in this microalga. This information and the identified target genes can now be used for metabolic engineering strategies towards tailored N. oleoabundans strains for industrial applications with increased lipid production and altered fatty acid composition.
2018-10-11 | GSE104807 | GEO
Project description:Non-alcoholic fatty liver disease (NAFLD) is characterized by excess lipid accumulation in hepatocytes and reprepresents a huge public health problem owing to its propensity to progress to non-alcoholic steatohepatitis (NASH), fibrosis, and liver failure. The lipids stored in hepatic steatosis are primarily triglycerides (TGs) synthesized by two acyl CoA:diacylglycerol acyltransferase (DGAT) enzymes. Either DGAT1 or DGAT2 catalyzes this reaction, and these enzymes have been suggested to differentially utilize exogenous or endogenously synthesized fatty acids, with DGAT2 being linked to storage of fatty acids from de novo lipogenesis, a process that is increased in NAFLD. However, whether DGAT2 is more responsible for lipid accumulation in NAFLD and the progression to fibrosis is currently unknown. Also, it is unresolved whether DGAT2 can be safely inhibited as a therapy for NAFLD. Here we induced NAFLD-like disease in mice by feeding a diet rich in fructose, saturated fat, and cholesterol and found that hepatocyte-specfici Dgat2 deficiency reduced expression of de novo lipogenesis genes and lowered liver TGs by ~70%. Importantly, the reduction of steatosis was not accompanied by increased inflammation or fibrosis, and insulin and glucose metabolism were unchanged. Conclusion: This study suggests that hepatic DGAT2 deficiency successfully reduced diet-induced hepatic steatosis and supports the development of DGAT2 inhibitors as a therapeutic strategy for treating NAFLD and preventing downstream consequences.
2020-05-26 | PXD013423 | Pride
Project description:The development of insulin resistance is strongly associated with accumulation of intracellular lipid in tissues outside of adipose including skeletal muscle, liver and heart. In obese humans, intramyocellular lipid (IMCL) is negatively correlated with whole body insulin sensitivity. The skeletal myocyte imports fatty acids (FA) into the cell from circulating free fatty acids or lipoprotein particles such as VLDL, to support energy production. Once transported into the cell, FAs are oxidized for ATP production, used to build membranes, or stored as triglyceride. However, in the long term, increased delivery of fatty acids can exceed mitochondrial oxidative capacity and set the stage for a vicious cycle of cellular lipotoxicity. We have recently identified a novel small molecule inhibitor of lipid accumulation in skeletal mycytes termed SBI-477. Microarray transcriptomics was performed in primary human skeletal myotubes following oleate loading and treatment with SBI-477. This was also compared to A922500, a diacylglycerol transferase 1 (DGAT1) inhibitor. SBI-477 treatment reversed many of the transcriptomic effects of oleate loading in these cells but also produced a transcriptomic profile distinct from the DGAT1 inhibitor. Microarray expression profiling was performed with total RNA isolated from differentiated primary human skeletal myotubes. These cells were treated with BSA (control for oleate), 100micromolar oleic acid, oleate + 10micromolar SBI-477 and oleate + 1micromolar A922500 for 24 hours. Oleate and test compound were added at the same time to the cells. Two biologic replicates for each condition were included.
2016-08-08 | E-GEOD-77212 | biostudies-arrayexpress
Project description:The development of insulin resistance is strongly associated with accumulation of intracellular lipid in tissues outside of adipose including skeletal muscle, liver and heart. In obese humans, intramyocellular lipid (IMCL) is negatively correlated with whole body insulin sensitivity. The skeletal myocyte imports fatty acids (FA) into the cell from circulating free fatty acids or lipoprotein particles such as VLDL, to support energy production. Once transported into the cell, FAs are oxidized for ATP production, used to build membranes, or stored as triglyceride. However, in the long term, increased delivery of fatty acids can exceed mitochondrial oxidative capacity and set the stage for a vicious cycle of cellular lipotoxicity. We have recently identified a novel small molecule inhibitor of lipid accumulation in skeletal mycytes termed SBI-477. Microarray transcriptomics was performed in primary human skeletal myotubes following oleate loading and treatment with SBI-477. This was also compared to A922500, a diacylglycerol transferase 1 (DGAT1) inhibitor. SBI-477 treatment reversed many of the transcriptomic effects of oleate loading in these cells but also produced a transcriptomic profile distinct from the DGAT1 inhibitor.
2016-08-08 | GSE77212 | GEO
Project description:Through scRNA-sequencing of primary human hepatocytes (PHHs), we have identified four subgroups of hepatocytes. A phenotyping 5-probe cocktail (Sanofi-Aventis) has been used to assess their metabolic capacity. Upon cocktail treatment, the characterized four hepatocyte subgroups displayed differential gene expression profiles and exhibited xenobiotic metabolism-related specialization. Intracellular lipid accumulation achieved through loading the cells with free fatty acids (FFA, 2:1 oleic:palmitic acid), differently affected the four subgroups. Moreover, we have shown that intracellular fat accumulation diminishes the drug-related metabolic capacity of hepatocytes.
2023-09-19 | E-MTAB-11530 | biostudies-arrayexpress
Project description:Abnormalities in hepatic lipid metabolism are believed to play a critical role in the etiology of nonalcoholic steatohepatitis (NASH). Monoacylglycerol acyltransferase (MGAT) enzymes convert monoacylglycerol to diacylglycerol, which is the penultimate step in one pathway for triacylglycerol (TAG) synthesis. Hepatic expression of Mogat1, which encodes an MGAT enzyme, is increased in the livers of mice with hepatic steatosis and knocking down Mogat1 improves insulin sensitivity, but whether increased MGAT activity plays a role in the etiology of NASH is unclear. To examine the effects of knocking down Mogat1 in the liver on the development of NASH, C57BL/6 mice were placed on a diet containing high levels of trans fatty acids, fructose, and cholesterol (HTF-C diet) or a low fat control diet for 4 weeks. Mice were then injected with antisense oligonucleotides (ASO) to knockdown Mogat1 or a scrambled ASO control for 12 weeks while remaining on diet. HTF-C diet caused glucose intolerance, hepatic steatosis, and induced hepatic gene expression markers of inflammation, macrophage infiltration, and stellate cell activation. Mogat1 ASO treatment, which suppressed Mogat1 expression in liver, attenuated weight gain, improved glucose tolerance, and decreased hepatic TAG content compared to control ASO-treated mice on HTF-C chow. However, Mogat1 ASO treatment did not reduce hepatic DAG, cholesterol, or free fatty acid content, improve histologic measures of liver injury, or reduce expression of markers of stellate cell activation, liver inflammation, and injury. In conclusion, inhibition of hepatic Mogat1 in HTF-C diet-fed mice improves glucose tolerance and hepatic TAG accumulation without attenuating liver inflammation and injury. Total RNA obtained from liver of 4 control vs. 4 Mogat1 ASO treated higf-fat diet (HFD) fed mice.
2014-11-05 | E-GEOD-60349 | biostudies-arrayexpress
Project description:When macrophages are activated by sensing bacterial lipopolysaccharides (LPS), they greatly increase their motility, mRNA synthesis and protein production. Most of the ATP needed for these responses is derived from the uptake and catabolism of glucose, a relatively inefficient ATP source. Although the stimulated cells also increase their uptake of free fatty acids, they store a large fraction as triglycerides (TAG). We report here that both Toll-like receptor 4 (TLR4) and TLR2 agonists stimulate prolonged TAG retention by primary murine and human macrophages. Agonist-induced TAG storage lasted at least 72-96 hrs in vitro and was associated with increases in fatty acid (FA) uptake, FA esterification, and FA incorporation into TAG; FA oxidation decreased. The results of expression and inhibitor studies support a prominent role for increases in long chain acyl CoA synthase 1 (ACSL1) and diacylglycerol acyltransferase-2 (DGAT2) during the sustained response to TLR2/4 activation; decreases in adipose triglyceride lipase (ATGL, Pnpla2) and monoacylglycerol lipase (MgII) may also contribute. Stimulated murine macrophages that retained TAG carried out phagocytosis more effectively and were protected from saturated fatty acid-induced cell death (lipotoxicity). TLR agonist-induced TAG retention in macrophages is thus an active, sustained process that may have important adaptive functions. It may also contribute to the persistence of lipid-laden macrophages in infected tissues, host susceptibility to some microbial pathogens, and the pathogenesis of atherosclerosis. RNA from macrophage loaded with Fatty Acids, stimulated with bacterial lipopolysaccharides (LPS), or both compared to untreated controls (FA, LPS, FA+LPS, untreated). Replicates from 4 independent experiments.
2013-12-17 | E-GEOD-42190 | biostudies-arrayexpress