CES1D Mouse

What is Ces1d and what distinguishes it from other carboxylesterases in mice?

Ces1d is a critical carboxylesterase enzyme that plays a central role in lipid metabolism, particularly in adipose tissue. It functions primarily as a lipid-digesting enzyme that produces a unique set of fatty acids essential for proper glucose and lipid metabolism . Among the Ces1 family members in mice (which includes Ces1a through Ces1g), Ces1d is particularly important as it represents the functional ortholog of human CES1 .

Ces1d can be distinguished from other mouse carboxylesterases by its tissue expression pattern and substrate specificity. It has been known by several aliases in the scientific literature, including Ces3, CesMH1, triacylglycerol hydrolase (TGH), and cholesteryl ester hydrolase (CEH) . Importantly, when studying Ces1d, researchers must use specific antibodies that can differentiate it from other Ces1 isoforms, as demonstrated in studies where anti-Ces1d antibodies (goat) specifically detected Ces1d but not other isoforms .

How does mouse Ces1d relate to human CES1 in structure and function?

Mouse Ces1d shares significant homology with human CES1, with 78% identity and 88% similarity at the amino acid level . This high degree of conservation suggests functional similarity between the species. Both proteins contain the HXEL ER retrieval sequences at their C-termini—specifically, mouse Ces1d contains HVEL while human CES1 contains HIEL . These retrieval sequences are critical for proper protein localization within the endoplasmic reticulum.

Functionally, both enzymes participate in similar metabolic processes, including lipid metabolism and drug disposition. Multiple studies have confirmed that Ces1d is the functional ortholog of human CES1 through comparative enzymatic and physiological analyses . This orthologous relationship makes Ces1d mouse models particularly valuable for translational research aimed at understanding human CES1 function in metabolic disorders.

What is the standard nomenclature system for carboxylesterases in research literature?

The nomenclature for carboxylesterases has been standardized to reduce confusion in the scientific literature. According to this system, mammalian carboxylesterases are grouped into five families based on homology and gene structure/chromosome localization . The capitalized "CES" root is used for human carboxylesterases, whereas "Ces" is used for mouse and rat carboxylesterases, followed by the family number and, if multiple genes exist in a family, a letter is added following the family number .

For genes, the nomenclature appears in italics (CES/Ces), while for proteins, non-italic notation (CES/Ces) is used. This standardization is crucial because incorrect ortholog assignments between mouse and human carboxylesterases have led to misinterpretations in previous studies . For example, mouse Ces1g was previously called Ces1, which incorrectly suggested it was the ortholog of human CES1, when the true functional ortholog is Ces1d .

How is Ces1d distributed across cellular compartments in adipose tissue?

Ces1d exhibits a unique distribution pattern in adipose tissue that changes under different metabolic conditions. While traditionally considered to be predominantly localized in the endoplasmic reticulum (ER), recent research has revealed a more complex distribution :

In adipose tissue, particularly under high-fat diet (HFD) conditions, a significant amount of Ces1d translocates from the ER onto lipid droplets. Immunofluorescence staining with anti-Ces1d and anti-PLIN1 (a lipid droplet marker protein) antibodies demonstrated colocalization of Ces1d with lipid droplets in subcutaneous white adipose tissue (sWAT) . This translocation appears to be tissue-specific, as in the liver, most Ces1d remains in the ER even under HFD conditions .

Subcellular fractionation experiments confirmed that significant amounts of Ces1d co-isolate with lipid droplets in sWAT, while remaining predominantly in the ER fraction in liver tissue . This differential distribution suggests distinct tissue-specific functions of Ces1d in lipid metabolism.

What mechanisms regulate Ces1d translocation between the ER and lipid droplets?

The translocation of Ces1d from the ER to lipid droplets represents a critical regulatory mechanism that appears to be influenced by metabolic state. Research has shown that under high-fat diet conditions, Ces1d levels decrease in the ER fraction while increasing in lipid droplet fractions in adipose tissue .

This translocation mechanism may involve several factors:

Understanding these translocation mechanisms is important for developing targeted interventions for metabolic disorders.

How can researchers generate tissue-specific Ces1d knockout mice for metabolic studies?

Researchers can generate adipose tissue-specific Ces1d knockout mice using the Cre-loxP system, which allows for targeted gene deletion in specific tissues. The methodology involves:

• Creating Ces1d floxed (Ces1d^flx/flx) mice: The Ces1d gene is flanked by loxP sites, which are recognition sequences for Cre recombinase .

• Breeding with tissue-specific Cre mice: The Ces1d^flx/flx mice are bred with transgenic mice expressing Cre recombinase under the control of a tissue-specific promoter. For adipose tissue-specific deletion, adiponectin-Cre mice are commonly used .

• Confirmation of knockout efficiency: Western blotting should be performed to confirm that Ces1d is efficiently ablated in the targeted tissue (WAT and BAT) but remains unaltered in other tissues such as the liver .

The resulting Fat-Ces1d knockout (FKO) mice can then be challenged with different diets, such as high-fat diet (HFD), to study the metabolic consequences of Ces1d deficiency specifically in adipose tissue . Littermate Ces1d^flx/flx mice without Cre expression are typically used as wild-type controls.

Alternatively, for complete Ces1 cluster knockout, CRISPR/Cas9 methodology can be employed to delete the entire Ces1 gene cluster, as demonstrated in the generation of Ces1^-/- mice . This approach involves coinjection of Cas9 mRNA, guide RNAs targeting flanking regions of the Ces1 cluster, and homologous recombination DNA oligonucleotides into zygotes .

What phenotypic characterization is essential when working with Ces1d knockout models?

Comprehensive phenotypic characterization of Ces1d knockout models should include:

• Body composition analysis:

  • Body weight monitoring over time

  • Fat mass and lean mass measurements using methods such as EchoMRI

  • Assessment of individual fat depot sizes (subcutaneous, epididymal, brown adipose tissue)

• Metabolic parameters:

  • Glucose tolerance and insulin sensitivity tests

  • Lipid profiles (triglycerides, cholesterol, fatty acids)

  • Energy expenditure measurements

• Histological examination:

  • H&E staining of adipose tissues to assess adipocyte morphology and lipid droplet size

  • Liver histology to evaluate hepatic steatosis

  • Immunohistochemistry for inflammatory markers

• Molecular analyses:

  • Expression analysis of genes involved in lipid metabolism

  • Assessment of mitochondrial function

  • Inflammation markers in adipose tissue and circulation

• Lipid droplet characterization:

  • Size and distribution of lipid droplets in adipocytes

  • Expression of lipid droplet-associated proteins

When characterizing Ces1d knockout mice, researchers should expect phenotypes such as increased body weight, larger fat mass, reduced lean mass, larger lipid droplet sizes in adipose tissues, and development of fatty liver when challenged with HFD .

How should researchers differentiate Ces1d from other Ces1 isoforms in experimental procedures?

Differentiating Ces1d from other Ces1 isoforms is critical for accurate experimental results. Researchers should employ the following approaches:

• Antibody selection: Use isoform-specific antibodies that have been validated for selectivity. For example, research has demonstrated the use of an anti-Ces1d antibody (goat) that specifically detects Ces1d but not other isoforms, compared to another antibody (mouse) that recognizes multiple Ces1 isoforms .

• Validation of antibody specificity: When introducing a new antibody, validate its specificity by testing on samples where Ces1d is known to be present or absent. Comparison of antibody recognition patterns between different tissues can help confirm specificity, as some isoforms may be tissue-restricted .

• Western blot analysis: When performing Western blots, the pattern of bands can help distinguish Ces1d from other isoforms. For instance, in adipose tissue, bands recognized by Ces1d-specific and pan-Ces1 antibodies completely merged (appearing as a single yellow color in dual-color detection systems), whereas in liver they only partially merged due to the presence of other Ces1 isoforms .

• PCR primers: Design primers that specifically amplify Ces1d and not other Ces1 family members. This is particularly important for gene expression analyses .

• Consider tissue distribution: Remember that the distribution of Ces1 isoforms varies by tissue. For example, some Ces1 isoforms exist in the liver but not in adipose tissue, which can be useful for distinguishing Ces1d in different experimental contexts .

How does Ces1d influence glucose metabolism and insulin sensitivity?

Ces1d plays a significant role in glucose metabolism and insulin sensitivity, primarily through its effects on lipid handling in adipose tissue. Research using adipose tissue-specific Ces1d knockout (FKO) mice has revealed:

• Impaired glucose metabolism: FKO mice exhibit deteriorated glucose tolerance and develop systemic insulin resistance when challenged with a high-fat diet .

• Mechanistic insights: Ces1d digests lipids in adipose tissue to produce a unique set of fatty acids that are essential for proper glucose metabolism . In the absence of Ces1d, this process is disrupted, leading to metabolic dysfunction.

• Secondary effects: The abnormal lipid handling in adipose tissue of FKO mice leads to ectopic accumulation of triglycerides in peripheral tissues, which is associated with insulin resistance .

• Clinical relevance: In human populations, the expression levels of CES1 (human homolog of Ces1d) are significantly higher in obese individuals with type 2 diabetes compared to obese individuals with normal glucose tolerance, suggesting a potential compensatory mechanism in response to metabolic stress .

• Tissue crosstalk: Ces1d deficiency in adipose tissue leads to secondary effects in other tissues, including exacerbated liver steatosis, which further contributes to systemic insulin resistance .

These findings highlight Ces1d as a key metabolic regulator that protects against diet-induced obesity and associated metabolic disorders, making it a potential therapeutic target for obesity-related conditions .

What is the role of Ces1d in lipid droplet regulation in adipocytes?

Ces1d plays a critical role in lipid droplet regulation in adipocytes, affecting both their size and function:

• Lipid droplet size control: Adipose tissue-specific Ces1d knockout (FKO) mice develop abnormally large lipid droplets in their adipocytes when challenged with a high-fat diet . This phenotype was observed across multiple adipose depots, including subcutaneous WAT, epidydimal WAT, and brown adipose tissue .

• Lipolytic activity: Ces1d has been reported to participate in basal lipolysis in adipose tissue, helping maintain normal lipid droplet size and preventing excessive lipid accumulation . Its unique localization on lipid droplets positions it to directly regulate lipid droplet metabolism.

• Lipid droplet-associating capacity: Under both normal and high-fat diet conditions, significant amounts of Ces1d translocate onto or in proximity to lipid droplets, as demonstrated by colocalization with PLIN1, a lipid droplet marker protein . This localization is critical for its function in lipid droplet metabolism.

• Tissue-specific action: While Ces1d associates with lipid droplets in adipose tissue, it remains predominantly in the ER in liver tissue, suggesting tissue-specific regulation of lipid droplets .

• Consequence of dysregulation: The enlarged lipid droplets observed in Ces1d-deficient adipocytes contribute to adipose tissue dysfunction and lead to ectopic lipid accumulation in other tissues like the liver, promoting systemic metabolic abnormalities .

Understanding Ces1d's role in lipid droplet regulation provides insights into fundamental mechanisms of adipocyte biology and the pathogenesis of obesity-associated metabolic disorders.

How can Ces1d research inform potential therapeutic strategies for metabolic disorders?

Ces1d research presents several promising avenues for therapeutic development targeting metabolic disorders:

• Target validation: Studies showing that Ces1d protects against diet-induced obesity and metabolic dysfunction validate it as a potential therapeutic target . The finding that Ces1d-deficient mice develop more severe metabolic phenotypes under high-fat diet conditions suggests that enhancing Ces1d activity might be beneficial in obesity and related disorders.

• Mechanism-based drug design: Understanding that Ces1d produces specific fatty acids essential for proper glucose and lipid metabolism provides a mechanistic basis for drug development . Compounds that mimic these fatty acids or enhance Ces1d activity could potentially improve metabolic health.

• Tissue-specific targeting: The observation that Ces1d translocation to lipid droplets in adipose tissue is critical for its function suggests that therapeutics specifically targeting this translocation process might be effective . This tissue-specific approach could minimize off-target effects in other tissues where Ces1d functions differently.

• Personalized medicine applications: Human data showing that CES1 (human homolog of Ces1d) levels are significantly increased in obese patients with type 2 diabetes compared to obese patients without diabetes suggests potential for stratifying patients based on CES1 expression or activity . This could inform personalized therapeutic approaches.

• Drug metabolism considerations: Research on Ces1 cluster knockout mice demonstrates their altered metabolism of drugs like irinotecan and capecitabine . This knowledge is crucial for predicting drug-drug interactions and optimizing dosing in patients with different metabolic profiles, particularly those with obesity or diabetes who may have altered Ces1d/CES1 expression.

What are the unique methodological challenges in studying Ces1d translocation to lipid droplets?

Studying Ces1d translocation to lipid droplets presents several methodological challenges that researchers must address:

• Isolation of intact lipid droplets: Obtaining pure lipid droplet fractions without contamination from other cellular compartments requires specialized protocols. Researchers must carefully validate their fractionation methods using markers for lipid droplets (e.g., PLIN1) as well as other compartments (e.g., PDI and Rtn4α for ER, ERK for cytosol) .

• Distinguishing Ces1d from other Ces1 isoforms: As multiple Ces1 isoforms exist and may have different localization patterns, researchers must use isoform-specific antibodies for immunofluorescence staining and Western blotting . This is particularly important given the historical confusion in carboxylesterase nomenclature and ortholog assignments.

• Live-cell imaging limitations: Tracking Ces1d translocation in real-time presents technical challenges due to the dynamic nature of lipid droplets and the need for fluorescent tagging that doesn't interfere with Ces1d function or localization.

• Quantifying translocation: Developing reliable methods to quantify the relative amounts of Ces1d in different cellular compartments is essential for understanding the regulation of translocation. This may require combining immunofluorescence with advanced imaging techniques such as super-resolution microscopy .

• Recreating physiological conditions: The translocation of Ces1d to lipid droplets appears to be influenced by metabolic state (e.g., high-fat diet) . Therefore, experimental systems must accurately recreate these physiological conditions to study translocation meaningfully.

• Distinguishing causes from consequences: Determining whether Ces1d translocation is a cause or consequence of metabolic changes requires careful experimental design, potentially involving inducible systems or time-course studies during metabolic challenges .

How do post-translational modifications affect Ces1d function and localization?

Post-translational modifications of Ces1d play important roles in regulating its function and localization, though our understanding remains incomplete:

Understanding the post-translational regulation of Ces1d is crucial for developing strategies to modulate its activity and localization for therapeutic purposes in metabolic disorders.