CES1G Mouse

What is Ces1g in mice and how does it relate to human carboxylesterases?

Ces1g (also known as Es-x) is a member of the carboxylesterase family in mice that plays significant roles in lipid metabolism. It's important to note that while Ces1g was previously referred to simply as Ces1, it is not the functional ortholog of human CES1. The functional mouse ortholog of human CES1 has been demonstrated to be Ces1d, not Ces1g . The functional human ortholog for Ces1g has not yet been definitively established, which creates an important consideration when attempting to translate findings between mouse models and human applications.
Carboxylesterases are a family of enzymes that hydrolyze esters, amides, and thioesters. In mice, there are multiple Ces genes categorized into different families, with Ces1g belonging to the Ces1 family that includes Ces1a through Ces1g, each with distinct substrate specificities and tissue distribution patterns .

What are the main physiological roles of Ces1g in mice?

Ces1g plays crucial roles in lipid metabolism and homeostasis. Research has demonstrated that Ces1g is involved in:

• Regulation of plasma cholesterol and triglyceride levels

• Intestinal cholesterol and fat absorption processes

• Macrophage cholesterol efflux

• Expression of lipid transport proteins including Niemann-Pick C1 like 1

• Regulation of macrophage polarization toward an M2 phenotype
  Global inactivation of Ces1g in mice leads to reduced plasma cholesterol levels, suggesting its importance in cholesterol metabolism . Ces1g also appears to influence the expression of ATP-binding cassette subfamily A member 1 (ABCA1) and ABCG1, which are critical for cholesterol efflux from macrophages .

How is Ces1g expression regulated in mouse tissues?

Ces1g expression appears to be regulated by multiple factors, with the Farnesoid X Receptor (FXR) playing a particularly important role. Studies suggest that Ces1g is a direct transcriptional target of FXR and may be involved in the regulation of liver lipid homeostasis by this nuclear receptor .
In FXR knockout mice, alterations in Ces1g expression contribute to changes in lipid metabolism, potentially influencing the development of metabolic disorders such as Metabolic-Associated Fatty Liver Disease (MAFLD) . The regulation of Ces1g may also involve other metabolic pathways, as suggested by transcriptomic analyses comparing FXR-knockout mice and human MAFLD samples .

What are the recommended methods for generating Ces1g knockout mice?

When generating Ces1g knockout mice, researchers have successfully employed several approaches:

• Conventional knockout strategies using homologous recombination

• CRISPR/Cas9 system for targeted gene disruption
  The CRISPR/Cas9 system has been effectively used to generate FXR knockout mice as described in the literature . A similar approach can be applied to Ces1g. When designing CRISPR guides, researchers should carefully consider potential off-target effects and confirm knockout efficiency through multiple validation methods, including:

• Genomic PCR and sequencing to confirm the deletion or mutation

• RT-qPCR to verify absence of mRNA transcripts

• Western blotting to confirm protein absence

• Functional assays to demonstrate loss of Ces1g enzymatic activity

How should researchers design experiments to study Ces1g's role in atherosclerosis?

When designing experiments to investigate Ces1g's role in atherosclerosis, researchers should consider the following methodological approaches:

How do Ces1g-deficient mice respond to different dietary challenges?

Ces1g-deficient mice exhibit differential responses to dietary challenges, which provides valuable insights into the gene's metabolic functions:
When challenged with a Western diet (high fat, high cholesterol), Ces1g-/- Ldlr-/- double knockout mice demonstrate:

• Decreased plasma cholesterol and triglyceride levels compared to Ldlr-/- controls

• Reduced development of atherosclerotic lesions

• Altered intestinal lipid absorption profiles

• Changes in cholesterol efflux from macrophages
  These dietary challenge studies reveal that Ces1g deficiency provides protection against diet-induced metabolic disorders, particularly by:

• Inhibiting intestinal cholesterol and fat absorption

• Reducing the expression of Niemann-Pick C1 like 1, a key cholesterol transporter

• Increasing macrophage cholesterol efflux through upregulation of ABCA1 and ABCG1

• Promoting M2 macrophage polarization

• Inducing the expression of hepatic cholesterol 7α-hydroxylase and sterol 12α-hydroxylase

What are the contradictory findings regarding Ces1g's role in atherosclerosis models?

An intriguing contradiction exists in the literature regarding Ces1g's role in atherosclerosis depending on the model system used:
Global Ces1g knockout: In Ldlr-/- background, global Ces1g deficiency is atheroprotective, leading to:

• Reduced plasma lipids

• Decreased atherosclerotic lesion development

• Enhanced macrophage cholesterol efflux

• Inhibited intestinal cholesterol absorption
  Liver-specific Ces1g knockdown: In contrast, when Ces1g is specifically knocked down in the liver of ApoE-/- mice, the opposite effect occurs:

• Increased hyperlipidemia

• Exacerbated Western diet-induced atherogenesis
  This contradiction highlights the complex, tissue-specific roles of Ces1g in lipid metabolism. The differential effects may be explained by:

• Tissue-specific functions of Ces1g in liver versus intestine

• Compensatory mechanisms that may be activated in global knockout but not in tissue-specific knockdown

• Potential differences in background strain interactions (Ldlr-/- versus ApoE-/-)

• Differences between developmental knockout versus adult knockdown approaches
  Researchers must carefully consider these contradictions when designing experiments and interpreting results from Ces1g mouse models.

How does Ces1g interact with nuclear receptors in metabolic regulation?

Ces1g appears to be intimately connected with nuclear receptor signaling, particularly with the Farnesoid X Receptor (FXR) pathway:
Studies suggest that Ces1g is a direct target of FXR and is involved in the regulation of liver lipid homeostasis controlled by this nuclear receptor . When FXR is deleted in mice (FXR-KO), significant hepatic steatosis develops when mice are challenged with a high-fat diet, with increased fat droplet ballooning compared to wild-type mice .
The interplay between Ces1g and FXR may occur through several mechanisms:

• Direct transcriptional regulation of Ces1g by FXR

• Ces1g-mediated alterations in bile acid metabolism, which in turn affects FXR activation

• Shared involvement in metabolic pathways related to fatty liver disease
  Interestingly, while FXR knockout exacerbates steatosis in mice, human studies have shown that FXR expression is not significantly different between MAFLD patients and controls . This suggests that changes in FXR protein activity, rather than expression, may be important, or that Ces1g may be regulated by additional factors beyond FXR in humans.

What approaches are recommended for analyzing transcriptomic changes in Ces1g-deficient models?

For comprehensive transcriptomic analysis of Ces1g-deficient models, researchers should consider the following methodological approaches:

• RNA isolation and quality control:

  • Extract total RNA from relevant tissues (liver, intestine, adipose)

  • Assess RNA quality using bioanalyzer (aim for RIN > 8)

  • Use appropriate RNA extraction methods depending on tissue type

• RNA sequencing considerations:

  • Use paired-end sequencing for better transcript reconstruction

  • Aim for >20 million reads per sample for adequate coverage

  • Include appropriate biological replicates (minimum n=3 per group)

• Bioinformatic analysis pipeline:

  • Map trimmed reads to reference genome (mouse GRCm38/mm10)

  • Perform differential expression analysis using established tools (DESeq2, edgeR)

  • Use stringent statistical thresholds (adjusted p-value < 0.05)

  • Validate key findings with RT-qPCR

• Pathway analysis strategies:

  • Perform Gene Ontology (GO) enrichment analysis

  • Conduct Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis

  • Use protein-protein interaction (PPI) analysis with STRING and Cytoscape

  • Apply molecular complex detection (MCODE) algorithm to identify densely connected network components

• Integration with public datasets:

  • Compare findings with publicly available datasets (e.g., GEO database)

  • Look for overlapping differentially expressed genes between mouse models and human samples

  • Focus on genes consistently altered across different datasets
    The research by Ma et al. illustrates the power of this approach, identifying 134 overlapping genes between FXR-KO mice and MAFLD patients, with enrichment in metabolic pathways, retinol metabolism, oxidation-reduction processes, and lipid metabolic processes .

How can researchers validate key gene expression changes in Ces1g models?

Validation of gene expression changes identified through transcriptomic analysis requires a multi-faceted approach:

• RT-qPCR validation:

  • Design primers spanning exon-exon junctions to avoid genomic DNA amplification

  • Use appropriate reference genes (validate stability across experimental conditions)

  • Include technical and biological replicates

  • Present data as fold-change relative to control group

• Protein-level validation:

  • Western blotting using validated antibodies

  • Immunohistochemistry to assess tissue distribution and localization

  • Consider using BCA protein assay kit for accurate protein quantification

• Functional validation:

  • Use siRNA knockdown in cellular models to confirm gene functions

  • For lipid metabolism genes, employ lipid droplet staining with BODIPY 493/503

  • Consider fluorescence-activated cell sorting (FACS) to isolate cells with differential expression

• Histological validation:

  • Perform H&E staining to assess histological changes

  • Use immunohistochemistry with protein-specific antibodies

  • For steatosis assessment, employ specialized lipid stains

• Translational validation:

  • Compare findings with human datasets (e.g., GSE48452, GSE63067)

  • Focus on genes that show consistent changes across species

  • Prioritize genes with established roles in relevant metabolic pathways

What are the implications of Ces1g research for understanding human metabolic disease?

Research on Ces1g mouse models provides several important insights into human metabolic diseases:

• The functional human ortholog for Ces1g has not been definitively established

• Human carboxylesterase biology may differ significantly from mouse

• Complex tissue-specific roles may require targeted therapeutic approaches

What are the current limitations in Ces1g mouse model research and how might they be addressed?

Current Ces1g mouse model research faces several important limitations that researchers should consider:

• Orthology uncertainty:

  • The functional human ortholog for Ces1g remains undefined

  • Research should focus on identifying the human equivalent to better translate findings

  • Comparative genomics and functional studies across species could help resolve this issue

• Strain background effects:

  • Different mouse strains may show variable Ces1g functions and phenotypes

  • Studies should be replicated across multiple genetic backgrounds

  • Backcrossing to congenic backgrounds may help standardize results

• Tissue-specific contributions:

  • The contradictory findings between global and liver-specific Ces1g manipulation highlight the need for comprehensive tissue-specific studies

  • Development of conditional knockout models for specific tissues (intestine, adipose, macrophage) would help resolve tissue-specific roles

• Sex-specific differences:

  • Many studies use only male mice, potentially missing sex-specific effects

  • Future research should include both sexes and analyze results separately

  • Hormonal influences on Ces1g expression and function should be investigated

• Diet composition variability:

  • Different studies use varied "Western diet" or "high-fat diet" compositions

  • Standardization of diet compositions or systematic testing of different diets would improve comparability between studies

  • Time-course studies would help understand adaptive responses to dietary challenges