CES2E Mouse

What is CES2E and how does it relate to other Ces2 family members in mice?

CES2E (Ces2e) is one of seven full-length carboxylesterase 2 genes identified in mice, alongside Ces2a, Ces2b, Ces2c, Ces2f, Ces2g, and Ces2h (with Ces2d being a pseudogene) . CES2E belongs to the carboxylesterase family that functions primarily as lipid hydrolases with significant activity against diglycerides (DG) and monoglycerides (MG).

Recent research demonstrates that CES2/Ces2 proteins are highly efficient diglyceride hydrolase (DGH) and monoglyceride hydrolase (MGH) enzymes that may play critical roles in liver and gut lipid signaling . This characteristic challenges earlier assumptions that there exists a single murine ortholog of human CES2, as multiple Ces2 family members appear to share functional similarities .

What is the tissue expression pattern of Ces2e in mice?

In wild-type mice, Ces2 genes, including Ces2e, are highly expressed in the small intestine. Ces2a-2e are also expressed quite abundantly in the liver, although their hepatic expression levels are not as high as some Ces1 genes . Quantitative real-time PCR (qRT-PCR) analyses have confirmed this distribution pattern.

Specifically, while Ces1 genes (especially Ces1b-1g) demonstrate predominant expression in the liver, Ces2 genes show stronger expression in intestinal tissues, with Ces2a-2e being particularly abundant in the small intestine . This tissue-specific expression pattern suggests Ces2e may have specialized functions in intestinal lipid metabolism.

What techniques are used to study Ces2e expression and activity?

Multiple complementary techniques are employed to comprehensively study Ces2e:

Expression Analysis:

• Real-time PCR using Ces2e-specific primers for mRNA quantification

• Western blotting of crude membrane fractions using specific antibodies

• Immunohistochemical staining for cellular and subcellular localization

Activity Assessment:

• Enzyme activity assays using specific lipid substrates to measure diglyceride hydrolase (DGH) and monoglyceride hydrolase (MGH) activities

• Pharmacokinetic studies with known CES2 substrate drugs to assess metabolic capacity

For gene expression studies, researchers have successfully employed RNeasy Mini Kit for RNA isolation from tissues, followed by cDNA synthesis using Maxima First Strand cDNA Synthesis Kit, and qRT-PCR with gene-specific primers . Western blotting of crude membrane fractions from different tissues provides protein-level confirmation, while immunohistochemistry offers insights into the cellular distribution of the enzyme .

How can researchers generate Ces2e knockout or modified mice?

Current approaches to Ces2e genetic manipulation include:

Ces2 Cluster Knockout:
Researchers have successfully generated complete Ces2 cluster knockout mice using CRISPR-Cas9 technology. The strategy involved designing guide RNAs targeting sequences flanking the entire Ces2 gene cluster, resulting in deletion of all functional Ces2 genes (Ces2a-2c and Ces2e-2h) .

Ces2e-Specific Modification:
For Ces2e-specific manipulation, researchers would need to design guide RNAs targeting sequences unique to the Ces2e gene while avoiding other Ces2 family members. Confirmation of specific Ces2e deletion requires PCR with gene-specific primers and sequencing to verify the precise genetic modification.

Humanized Models:
Researchers have developed humanized mice with stable transgenic human CES2 expression in a Ces2 cluster knockout background. Two approaches have been documented:

• Liver-predominant expression using ApoE promoter with hepatic control region (HCR)

• Intestine-predominant expression using Villin promoter

What roles does Ces2e play in lipid metabolism?

Ces2e, along with other Ces2 family members, exhibits high diglyceride hydrolase (DGH) and monoglyceride hydrolase (MGH) activities, suggesting important functions in lipid metabolism and signaling pathways . Research indicates that intestine-specific Ces2c overexpression can counteract diet-induced obesity and non-alcoholic fatty liver disease (NAFLD) in mice, potentially by increasing chylomicron particle size and accelerating chylomicron lipid uptake in muscles .

While the specific contribution of Ces2e to lipid metabolism hasn't been fully distinguished from other Ces2 family members in the available research, its enzymatic activity profile suggests similar potential for modulating lipid homeostasis. The high expression of Ces2e in both intestine and liver positions it as a potentially important regulator of lipid processing in these metabolically active tissues.

How does Ces2e contribute to drug metabolism?

Ces2 family proteins, including Ces2e, significantly impact the metabolism of certain pharmaceutical compounds. In Ces2 cluster knockout mice, researchers observed:

• A 5.3-fold greater plasma AUC0-2h of capecitabine compared to wild-type mice, indicating reduced drug clearance

• Dramatically higher capecitabine concentrations in the liver (13.8-fold) and small intestine (10.6-fold) of knockout mice compared to wild-type

• Significantly reduced conversion of vinorelbine to deacetylvinorelbine, with the deacetylvinorelbine-to-vinorelbine ratio in liver decreasing 333-fold in Ces2 knockout mice

How does mouse Ces2e compare to human CES2?

Research challenges the earlier notion that a single murine Ces2 gene (previously thought to be Ces2c) represents the ortholog of human CES2. Instead, multiple Ces2 proteins, including Ces2e, share functional similarities with human CES2 .

Key comparative aspects include:

• Both mouse Ces2e and human CES2 demonstrate efficient diglyceride hydrolase (DGH) and monoglyceride hydrolase (MGH) activities

• Mouse Ces2e, along with other Ces2 family members, appears functionally redundant in some aspects, whereas humans have a single CES2 gene

• The tissue distribution patterns show similarities, with expression in both liver and intestine

Functional complementation studies with humanized mice expressing human CES2 in either liver or intestine on a Ces2 cluster knockout background demonstrate that human CES2 can partially rescue the metabolic phenotypes observed in Ces2 knockout mice, suggesting conserved functionality .

What is the functional relationship between different Ces2 family members?

The Ces2 gene family in mice exhibits considerable functional redundancy, with multiple members showing similar enzymatic activities. All investigated CES2/Ces2 proteins demonstrate high activity as triglyceride hydrolases (TGH), diglyceride hydrolases (DGH), and monoglyceride hydrolases (MGH) .

This functional overlap complicates the study of individual Ces2 members, including Ces2e. The genetic approach of deleting the entire Ces2 cluster provides insights into the collective function of Ces2 proteins but requires complementary strategies to delineate member-specific roles.

Interestingly, while Ces2 cluster deletion dramatically affects the metabolism of certain drugs, it doesn't appear to significantly alter other Ces carboxylesterase family members' expression. Research showed no significant changes in Ces1 gene expression in Ces2 knockout mice, though there was a decrease in Ces3a and Ces3b levels in the small intestine .

How can Ces2e mouse models advance drug development research?

Ces2e and other Ces2 family members significantly impact drug metabolism, making Ces2 mouse models valuable tools for pharmaceutical research:

Predictive Pharmacokinetics:
Ces2 cluster knockout and humanized CES2 mice provide complementary models to predict drug metabolism differences between species. Researchers can utilize these models to:

• Evaluate the metabolic fate of ester-containing drug candidates

• Identify potential species differences in drug clearance

• Assess tissue-specific drug activation or inactivation

Drug-Drug Interaction Studies:
These mouse models enable investigation of potential drug-drug interactions involving CES2 substrates. For example, researchers can examine how concomitant medications might alter the metabolism of CES2-dependent prodrugs.

What insights can Ces2e research provide for metabolic disorders?

Research on intestine-specific Ces2c overexpression has demonstrated protection against diet-induced obesity and non-alcoholic fatty liver disease (NAFLD) in mice . While Ces2e-specific interventions haven't been explicitly studied in the provided references, the shared enzymatic activities among Ces2 family members suggest potential metabolic benefits from Ces2e modulation.

Ces2e research may prove valuable for understanding and potentially treating metabolic disorders through several mechanisms:

• Modulation of lipid absorption and processing in the intestine

• Alteration of lipid signaling pathways in metabolically active tissues

• Changes in chylomicron formation and systemic lipid distribution

• Potential impacts on insulin sensitivity and glucose metabolism

Future studies utilizing Ces2e-specific genetic manipulations could provide more precise insights into the specific contributions of this enzyme to metabolic health and disease.

What controls should be included when studying Ces2e function?

Robust experimental design for Ces2e research should incorporate several essential controls:

Genetic Models:

• Wild-type mice as baseline controls

• Ces2 cluster knockout mice to eliminate all Ces2 activity

• Ces2e-specific knockout mice (when available) to isolate Ces2e effects

• Humanized CES2 mice to assess functional conservation

• Tissue-specific expression models to determine site-specific contributions

Expression Analysis:

• Multiple tissue sampling to account for differential expression

• Analysis of all Ces family members to detect compensatory changes

• Time-course studies to capture dynamic expression changes

• Inclusion of positive control tissues with known high expression

Enzymatic Assays:

• Substrate specificity controls using multiple lipid substrates

• Kinetic analyses to determine enzyme efficiency parameters

• Inhibitor studies to confirm specificity of observed activities

• Comparison with recombinant enzymes of known activity

How should researchers account for compensatory mechanisms in Ces2e studies?

When investigating Ces2e function through genetic manipulation, researchers must consider potential compensatory changes:

• Comprehensive Expression Profiling: Analyze expression of all Ces family members (Ces1, Ces2, and Ces3) in response to Ces2e manipulation .

• Temporal Assessment: Distinguish between acute responses to gene deletion and long-term adaptive changes by examining expression patterns at multiple time points.

• Tissue-Specific Analysis: Examine compensatory responses across multiple tissues, as the pattern may differ between liver, intestine, and other sites of Ces expression.

• Functional Redundancy Testing: Assess whether other enzymes with similar activities (within or outside the Ces family) show increased activity in response to Ces2e deficiency.

• Conditional Models: Consider using inducible knockout systems to distinguish between developmental compensation and functional adaptation in adult animals.

Research has shown that Ces2 cluster deletion does not significantly alter Ces1 gene expression but does decrease Ces3a and Ces3b levels in the small intestine, highlighting the importance of examining potential cross-family compensatory mechanisms .

How can Ces2e research inform human drug metabolism studies?

Ces2e research provides valuable insights into human drug metabolism through comparative and translational approaches:

Species Differences in Drug Processing:
Studies comparing wild-type mice, Ces2 knockout mice, and humanized CES2 mice reveal important differences in drug metabolism between species. For capecitabine, Ces2 deletion dramatically increased plasma and tissue drug concentrations, while humanized CES2 expression partially restored normal metabolism .

Predictive Models for Drug Development:
Researchers can leverage Ces2e studies to develop more accurate predictions of human drug metabolism, particularly for ester-containing compounds and prodrugs that require carboxylesterase-mediated activation.

What potential therapeutic targets might emerge from Ces2e research?

Ces2e research may identify novel therapeutic targets for metabolic and pharmacological interventions:

Metabolic Disease Treatment:
Given that intestinal Ces2c overexpression counteracts diet-induced obesity and NAFLD in mice , Ces2e modulation might represent a potential therapeutic approach for metabolic disorders. Targeting Ce2e activity could potentially alter lipid processing, storage, and signaling in ways that improve metabolic health.

Improving Drug Efficacy:
Understanding Ces2e's role in drug metabolism could enable the development of strategies to modulate its activity selectively. This approach might enhance the efficacy of certain prodrugs by controlling their activation or extend the half-life of active compounds by reducing their metabolism.

Tissue-Specific Interventions: The differential expression of Ces2e between liver and intestine offers opportunities for tissue-targeted therapeutic approaches, potentially reducing systemic side effects while maximizing local efficacy.