GFP and mCherry gene expression at single cell level of all liver cell types from B6J mice
Ontology highlight
ABSTRACT: Diacylglycerol acyltransferase (DGAT)-2 catalyzes the final step of triglyceride (TG) synthesis. DGAT2 deletion in mice lowers liver TGs and DGAT2 overexpression might have the opposite effect. Mouse DGAT2 was overexpressed in C57Bl/6J mice using adeno-associated virus 8 (AAV8 and AAV-DJ). The tissue specificity of AAV8 and AAV-DJ was first evaluated. To confirm that AAV8 and AAV-DJ only infected hepatocytes and not other cells in liver, we carried out single cell sequencing of liver cells isolated from mice infected with AAV8-mCherry and AAV-DJ-GFP. The mCherry and GFP signals were highly expressed in hepatocytes and not in stellate, endothelial, or Kupffer cells.
Project description:Analysis of changes in gene expression following hepatocyte specific deletion of GATA4 in adult mice. Results showed that the subset of differentially expressed genes had liver specific ontologies. Total RNA isolated from hepatocytes of AAV8-Tbg-Cre injected GATA4 fl/fl mice was compared to total RNA isolated from AAV-Tbg-GFP injected GATA4 fl/fl mice.
Project description:Analysis of changes in gene expression following hepatocyte specific deletion of GATA4 and GATA6 in adult mice. Results showed that the subset of differentially expressed genes had liver specific ontologies. Total RNA isolated from hepatocytes of AAV8-Tbg-Cre injected GATA4,6 fl/fl mice was compared to total RNA isolated from AAV-Tbg-GFP injected GATA4,6 fl/fl mice.
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.
Project description:We compared the effects of Notch1 and Notch2 signaling induced by AAV8-mediated forced expression of Notch1 intracellular domain (NICD1) and Notch2 intracellular domain (NICD2), respectively, in the liver. Eight-week-old wild-type male mice were injected intraperitoneally with an NICD1- and/or NICD2-overexpressing AAV8 vector. Efficient AAV8-mediated gene transfer was confirmed by co-expressed green fluorescent protein (GFP) fluorescence in hepatocytes. A comprehensive gene expression analysis using DNA microarray indicated that the expression levels of 1704 and 1325 genes were altered by overexpressing NICD1 and NICD2, respectively, compared with the control group expressing only GFP.
Project description:In zebrafish, maternally deposited yolk is the source of nutrients for embryogenesis prior to digestive system maturation. Yolk nutrients are processed and secreted to the growing organism by an extra-embryonic tissue, the yolk syncytial layer (YSL). Export of lipid from the YSL occurs through the production of triacylglycerol-rich lipoproteins. Here we report that mutations in the triacylglycerol synthesis enzyme, diacylglycerol acyltransferase-2 (Dgat2), cause yolk sac opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. Though triacylglycerol synthesis continues, it is not properly coupled to lipoprotein production as dgat2 mutants produce fewer, smaller, ApoB-containing lipoproteins. Unlike DGAT2-null mice, which are lipopenic and die soon after birth, zebrafish dgat2 mutants are viable, fertile and exhibit normal mass and adiposity. Residual Dgat activity cannot be explained by the activity of other known Dgat isoenzymes, as dgat1a;dgat1b;dgat2 triple mutants continue to produce YSL lipid droplets and remain viable as adults. Further, the newly identified diacylglycerol acyltransferase, Tmem68, is also not responsible for the residual triacylglycerol synthesis activity. Unlike overexpression of Dgat1a and Dgat1b, monoacylglycerol acyltransferase-3 (Mogat3b) overexpression does not rescue yolk opacity, suggesting it does not possess Dgat activity in the YSL. However, mogat3b;dgat2 double mutants exhibit increased yolk opacity and often have structural alterations of the yolk extension. Quadruple mogat3b;dgat1a;dgat1b;dgat2 mutants either have severely reduced viability and stunted growth, or do not survive past 3 days post fertilization, depending on the dgat2 mutant allele present. Our study highlights the remarkable ability of vertebrates to synthesize triacylglycerol through multiple biosynthetic pathways.
Project description:To investigate the effect of TRIB3 overexpression on regulation of lipid metabolism in hepatocytes, we isolated mouse primary hepatocytes from AAV-GFP or AAV-Trib3 mice. We then performed gene expression profiling analysis using data obtained from RNA-seq of two groups of mouse primary hepatocytes from AAV-GFP or AAV-Trib3 mice.
Project description:The organoids heterogenously expressed mCherry by the lentivirus transduction were dissociated into single cells. A single mCherry- positive and -negative cell were collected and grown up to the organoids, respectivery. The difference of the gene expression profile between mCherry- positive and -negative organoids was assessd by maicroarray.
Project description:Regulation of RNA processing contributes profoundly to tissue development and physiology. The serine-arginine-rich splicing factor 1 (SRSF1) is essential for hepatocyte function and survival. Although SRSF1 is mainly known for its many roles in mRNA metabolism, it is also crucial for maintaining genome stability. We show that acute liver damage in the setting of targeted SRSF1 deletion in mice is primarily mediated by the excessive formation of deleterious RNA–DNA hybrids (R-loops), which induce DNA damage. Combining hepatocyte-specific transcriptome, proteome, and RNA binding analyses, we demonstrate that widespread genotoxic stress following SRSF1 depletion results in global inhibition of mRNA transcription and protein synthesis, leading to impaired metabolism and trafficking of lipids. Accumulation of lipids in SRSF1-deficient hepatocytes is quickly followed by necroptotic cell death, inflammation, and fibrosis, resulting in NASH-like liver pathology. This pathogenesis is recapitulated in SRSF1-depleted human liver cancer cells illustrating a conserved and fundamental role for SRSF1 in preserving genome integrity and tissue homeostasis. This data set contains a proteomic comparison of hepatocytes from wild type vs. acute knockout of SRSF1. The acute knockout was generated by injecting 8-week-old SRSF1 fl/fl mice with a viral vector expressing Cre under the control of the liver-specific thyroxine binding globulin (TBG) promoter (AAV8-TBG-iCre). Controls were generated by injecting AAV8-TBG-GFP viral vector. The hepatocytes were isolated 2 weeks post injection.
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.
Project description:We report transcriptional profiles of liver from control and p21-AAV8 administered no-pregnant mice and pregnant mice at dpc8 and dpc16. We revealed that genes related to carbohydrate transport and regulators of glucose and glycogen metabolism were down regulated in AAV-p21 administered liver at dpc16.