Gene_locus Report for: mouse-Ces1d

Carboxylesterase 3 (Ces3) is a hydrolase with a wide range of activities in liver and adipose tissue. In this study, we identified Ces3 as a major lipid droplet surface-targeting protein in adipose tissue upon cold exposure by liquid chromatography-tandem mass spectrometry. To investigate the function of Ces3 in the beta-adrenergic signaling-activated adipocytes, we applied WWL229, a specific Ces3 inhibitor, or genetic inhibition by siRNA to Ces3 on isoproterenol (ISO)-treated 3T3-L1 and brown adipocyte cells. We found that blockage of Ces3 by WWL229 or siRNA dramatically attenuated the ISO-induced lipolytic effect in the cells. Furthermore, Ces3 inhibition led to impaired mitochondrial function measured by Seahorse. Interestingly, Ces3 inhibition attenuated an ISO-induced thermogenic program in adipocytes by downregulating Ucp1 and Pgc1alpha genes via peroxisome proliferator-activated receptor gamma. We further confirmed the effects of Ces3 inhibition in vivo by showing that the thermogenesis in adipose tissues was significantly attenuated in WWL229-treated or adipose tissue-specific Ces3 heterozygous knockout (Adn-Cre-Ces3(flx/wt)) mice. As a result, the mice exhibited dramatically impaired ability to defend their body temperature in coldness. In conclusion, our study highlights a lipolytic signaling induced by Ces3 as a unique process to regulate thermogenesis in adipose tissue.

Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means are often required to determine the biologically relevant target (or targets) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3, also known as Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is markedly elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets.

A novel murine cDNA for triacylglycerol hydrolase (TGH), an enzyme that is involved in mobilization of triacylglycerol from storage pools in hepatocytes, has been cloned and expressed. The cDNA consists of 1962 bp with an open reading frame of 1695 bp that encodes a protein of 565 amino acids. Murine TGH is a member of the CES1A class of carboxylesterases and shows a significant degree of identity to other carboxylesterases from rat, monkey and human. Expression of the cDNA in McArdle RH7777 hepatoma cells showed a 3-fold increase in the hydrolysis of p-nitrophenyl laurate compared to vector-transfected cells. The highest expression of TGH was observed in the livers of mice, with lower expression in kidney, heart, adipose and intestinal (duodenum/jejunum) tissues. The murine gene that encodes TGH was cloned and exon-intron boundaries were determined. The gene spans approx. 35 kb and contains 14 exons. The results will permit future studies on the function of this gene via gene-targeting experiments and analysis of transcriptional regulation of the TGH gene.

The mammalian carboxylesterase 1 (Ces1/CES1) family comprises several enzymes that hydrolyze many xenobiotic chemicals and endogenous lipids. To investigate the pharmacological and physiological roles of Ces1/CES1, we generated Ces1 cluster knockout (Ces1 (-/-) ) mice, and a hepatic human CES1 transgenic model in the Ces1 (-/-) background (TgCES1). Ces1 (-/-) mice displayed profoundly decreased conversion of the anticancer prodrug irinotecan to SN-38 in plasma and tissues. TgCES1 mice exhibited enhanced metabolism of irinotecan to SN-38 in liver and kidney. Ces1 and hCES1 activity increased irinotecan toxicity, likely by enhancing the formation of pharmacodynamically active SN-38. Ces1 (-/-) mice also showed markedly increased capecitabine plasma exposure, which was moderately decreased in TgCES1 mice. Ces1 (-/-) mice were overweight with increased adipose tissue, white adipose tissue inflammation (in males), a higher lipid load in brown adipose tissue, and impaired blood glucose tolerance (in males). These phenotypes were mostly reversed in TgCES1 mice. TgCES1 mice displayed increased triglyceride secretion from liver to plasma, together with higher triglyceride levels in the male liver. These results indicate that the carboxylesterase 1 family plays essential roles in drug and lipid metabolism and detoxification. Ces1 (-/-) and TgCES1 mice will provide excellent tools for further study of the in vivo functions of Ces1/CES1 enzymes.

Carboxylesterase 1d (Ces1d) is a crucial enzyme with a wide range of activities in multiple tissues. It has been reported to localize predominantly in ER. Here, we found that Ces1d levels are significantly increased in obese patients with type 2 diabetes. Intriguingly, a high level of Ces1d translocates onto lipid droplets where it digests the lipids to produce a unique set of fatty acids. We further revealed that adipose tissue-specific Ces1d knock-out (FKO) mice gained more body weight with increased fat mass during a high fat-diet challenge. The FKO mice exhibited impaired glucose and lipid metabolism and developed exacerbated liver steatosis. Mechanistically, deficiency of Ces1d induced abnormally large lipid droplet deposition in the adipocytes, causing ectopic accumulation of triglycerides in other peripheral tissues. Furthermore, loss of Ces1d diminished the circulating free fatty acids serving as signaling molecules to trigger the epigenetic regulations of energy metabolism via lipid-sensing transcriptional factors, such as HNF4alpha. The metabolic disorders induced an unhealthy microenvironment in the metabolically active tissues, ultimately leading to systemic insulin resistance.

Mouse carboxylesterase 1d (Ces1d) and its human ortholog CES1 have been described to possess lipase activity and play a role in hepatic triacylglycerol (TG) metabolism and very-low density lipoprotein (VLDL) assembly. Liver is a central organ regulating cholesterol synthesis, storage, transport, and elimination. It has been proposed that Ces1d/CES1 might also catalyze cholesteryl ester (CE) hydrolysis in the liver and be responsible for the hydrolysis of high-density lipoprotein (HDL)-derived CE, thus contributing to the final step in the reverse cholesterol transport (RCT) pathway, where cholesterol is secreted from the liver into bile and feces, either directly or after conversion to water-soluble bile salts. However, the proposed function of Ces1d/CES1 as a CE hydrolase is controversial. We interrogated the role hepatic Ces1d plays in cholesterol homeostasis using liver-specific Ces1d deficient mice. We rationalized that if Ces1d is a major hepatic CE hydrolase, its absence would 1) reduce in vivo RCT flux, and 2) provoke liver CE accumulation after high cholesterol diet challenge. In this study, liver-specific Ces1d KO mice did not show any difference in the flux of in vivo HDL-to-feces RCT and did not cause additional liver CE accumulation after high-fat, high cholesterol Western-type diet (WTD) feeding, thereby challenging the importance of Ces1d as a major hepatic CE hydrolase.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Triacylglycerol accumulation in the liver is a hallmark of NAFLD. Metabolic studies have confirmed that increased hepatic de novo lipogenesis (DNL) in humans contributes to fat accumulation in the liver and to NAFLD progression. Mice deficient in carboxylesterase (Ces)1d expression are protected from high-fat diet-induced hepatic steatosis. To investigate whether loss of Ces1d can also mitigate steatosis induced by over-activated DNL, WT and Ces1d-deficient mice were fed a lipogenic high-sucrose diet (HSD). We found that Ces1d-deficient mice were protected from HSD-induced hepatic lipid accumulation. Mechanistically, Ces1d deficiency leads to activation of AMP-activated protein kinase and inhibitory phosphorylation of acetyl-CoA carboxylase. Together with our previous demonstration that Ces1d deficiency attenuated high-fat diet-induced steatosis, this study suggests that inhibition of CES1 (the human ortholog of Ces1d) might represent a novel pharmacological target for prevention and treatment of NAFLD.

Carboxylesterase 3 (Ces3) is a hydrolase with a wide range of activities in liver and adipose tissue. In this study, we identified Ces3 as a major lipid droplet surface-targeting protein in adipose tissue upon cold exposure by liquid chromatography-tandem mass spectrometry. To investigate the function of Ces3 in the beta-adrenergic signaling-activated adipocytes, we applied WWL229, a specific Ces3 inhibitor, or genetic inhibition by siRNA to Ces3 on isoproterenol (ISO)-treated 3T3-L1 and brown adipocyte cells. We found that blockage of Ces3 by WWL229 or siRNA dramatically attenuated the ISO-induced lipolytic effect in the cells. Furthermore, Ces3 inhibition led to impaired mitochondrial function measured by Seahorse. Interestingly, Ces3 inhibition attenuated an ISO-induced thermogenic program in adipocytes by downregulating Ucp1 and Pgc1alpha genes via peroxisome proliferator-activated receptor gamma. We further confirmed the effects of Ces3 inhibition in vivo by showing that the thermogenesis in adipose tissues was significantly attenuated in WWL229-treated or adipose tissue-specific Ces3 heterozygous knockout (Adn-Cre-Ces3(flx/wt)) mice. As a result, the mice exhibited dramatically impaired ability to defend their body temperature in coldness. In conclusion, our study highlights a lipolytic signaling induced by Ces3 as a unique process to regulate thermogenesis in adipose tissue.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in developed countries. NAFLD describes a wide range of liver pathologies from simple steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. NASH is distinguished from simple steatosis by inflammation, cell death and fibrosis. In this study we found that mice lacking triacylglycerol hydrolase (TGH, also known as carboxylesterase 3 or carboxylesterase 1d) are protected from high-fat diet (HFD) - induced hepatic steatosis via decreased lipogenesis, increased fatty acid oxidation and improved hepatic insulin sensitivity. To examine the effect of the loss of TGH function on the more severe NAFLD form NASH, we ablated Tgh expression in two independent NASH mouse models, Pemt(-/-) mice fed HFD and Ldlr(-/-) mice fed high-fat, high-cholesterol Western-type diet (WTD). TGH deficiency reduced liver inflammation, oxidative stress and fibrosis in Pemt(-/-) mice. TGH deficiency also decreased NASH in Ldlr(-/-) mice. Collectively, these findings indicate that TGH deficiency attenuated both simple hepatic steatosis and irreversible NASH.

Phenotypic screening is making a comeback in drug discovery as the maturation of chemical proteomics methods has facilitated target identification for bioactive small molecules. A limitation of these approaches is that time-consuming genetic methods or other means are often required to determine the biologically relevant target (or targets) from among multiple protein-compound interactions that are typically detected. Here, we have combined phenotypic screening of a directed small-molecule library with competitive activity-based protein profiling to map and functionally characterize the targets of screening hits. Using this approach, we identify carboxylesterase 3 (Ces3, also known as Ces1d) as a primary molecular target of bioactive compounds that promote lipid storage in adipocytes. We further show that Ces3 activity is markedly elevated during adipocyte differentiation. Treatment of two mouse models of obesity-diabetes with a Ces3 inhibitor ameliorates multiple features of metabolic syndrome, illustrating the power of the described strategy to accelerate the identification and pharmacologic validation of new therapeutic targets.

OBJECTIVE: Liver is the major organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or as bile acids. Intracellular hydrolysis of lipoprotein-derived cholesteryl esters (CEs) is essential to generate the free cholesterol required for this process. Earlier, we demonstrated that overexpression of human CE hydrolase (Gene symbol CES1) increased bile acid synthesis in human hepatocytes and enhanced reverse cholesterol transport in mice. The objective of the present study was to demonstrate that liver-specific deletion of its murine ortholog, Ces3, would decrease cholesterol elimination from the body and increase atherosclerosis. APPROACH AND RESULTS: Liver-specific Ces3 knockout mice (Ces3-LKO) were generated, and Ces3 deficiency did not affect the expression of genes involved in cholesterol homeostasis and free cholesterol or bile acid transport. The effects of Ces3 deficiency on the development of Western diet-induced atherosclerosis were examined in low density lipoprotein receptor knock out(-/-) mice. Despite similar plasma lipoprotein profiles, there was increased lesion development in low density lipoprotein receptor knock out(-/-)Ces3-LKO mice along with a significant decrease in the bile acid content of bile. Ces3 deficiency significantly reduced the flux of cholesterol from [(3)H]-CE-labeled high-density lipoproteins to feces (as free cholesterol and bile acids) and decreased total fecal sterol elimination. CONCLUSIONS: Our results demonstrate that hepatic Ces3 modulates the hydrolysis of lipoprotein-delivered CEs and thereby regulates free cholesterol and bile acid secretion into the feces. Therefore, its deficiency results in reduced cholesterol elimination from the body, leading to significant increase in atherosclerosis. Collectively, these data establish the antiatherogenic role of hepatic CE hydrolysis.

RATIONALE: Carboxylesterase 3/triacylglycerol hydrolase (TGH) has been shown to participate in hepatic very low-density lipoprotein (VLDL) assembly. Deficiency of TGH in mice lowers plasma lipids and atherogenic lipoproteins without inducing hepatic steatosis. OBJECTIVE: To investigate the contribution of TGH to atherosclerotic lesion development in mice that lack low-density lipoprotein receptor (LDLR). METHODS AND
RESULTS: Mice deficient in LDL receptor (Ldlr(-/-)) and mice lacking both TGH and LDLR (Tgh(-/-)/Ldlr(-/-)) were fed with a Western-type diet for 12 weeks. Analysis of Tgh(-/-)/Ldlr(-/-) plasma showed an atheroprotective lipoprotein profile with decreased cholesterol in the VLDL and the LDL fractions, concomitant with elevated high-density lipoprotein cholesterol. Significantly reduced plasma apolipoprotein B levels were also observed in Tgh(-/-)/Ldlr(-/-) mice. Consequently, Tgh(-/-)/Ldlr(-/-) mice presented with a significant reduction (54%, P<0.01) of the high-fat, high-cholesterol dieteninduced atherosclerotic plaques when compared with Tgh(+/+)/Ldlr(-/-) mice in the cross-sectional aortic root analysis. TGH deficiency did not further increase liver steatosis despite lowering plasma lipids, mainly due to reduced hepatic lipogenesis. The ameliorated dyslipidemia in Tgh(-/-)/Ldlr(-/-) mice was accompanied with significantly improved insulin sensitivity.
CONCLUSIONS: Inhibition of TGH activity ameliorates atherosclerosis development and improves insulin sensitivity in Ldlr(-/-) mice.

Previous work demonstrated both acid and neutral, bile salt-independent retinyl ester hydrolase activities in rat liver homogenates. Here we present the purification, identification, and characterization of an acid retinyl ester hydrolase activity from solubilized rat liver microsomes. Purification to homogeneity was achieved by sequential chromatography using SP-Sepharose cation exchange, phenyl-Sepharose hydrophobic interaction, concanavalin A-Sepharose affinity and Superose 12 gel filtration chromatography. The isolated protein had a monomer molecular mass of approximately 62 kDa, as measured by mass spectrometry. Gel filtration chromatography of the purified protein revealed a native molecular mass of approximately 176 kDa, indicating that the protein exists as a homotrimeric complex in solution. The purified protein was identified as carboxylesterase ES-10 (EC 3.1.1.1) by N-terminal Edman sequencing and extensive LC-MS/MS sequence analysis and cross-reaction with an anti-ES-10 antibody. Glycosylation analysis revealed that only one of two potential N-linked glycosylation sites is occupied by a high mannose-type carbohydrate structure. Using retinyl palmitate in a micellar assay system the enzyme was active over a broad pH range and displayed Michaelis-Menten kinetics with a K(m) of 86 microm. Substrate specificity studies showed that ES-10 is also able to catalyze hydrolysis of triolein. Cholesteryl oleate was not a substrate for ES-10 under these assay conditions. Real time reverse transcriptase-PCR and Western blot analysis revealed that ES-10 is highly expressed in liver and lung. Lower levels of ES-10 mRNA were also found in kidney, testis, and heart. A comparison of mRNA expression levels in liver demonstrated that ES-10, ES-4, and ES-3 were expressed at significantly higher levels than ES-2, an enzyme previously thought to play a major role in retinyl ester metabolism in liver. Taken together these data indicate that carboxylesterase ES-10 plays a major role in the hydrolysis of newly-endocytosed, chylomicron retinyl esters in both neutral and acidic membrane compartments of liver cells.

Several mouse carboxylesterase (CES) isozymes have been identified, but information about their roles in drug metabolism is limited. In this study, we purified and characterized a mouse CES1 isozyme that was induced by di-(2-ethylhexyl) phthalate. Purified mouse CES1 shared some biological characteristics with other CES isozymes, such as molecular weight of a subunit and isoelectronic point. In addition, purified mouse CES1 behaved as a trimer, a specific characteristic of CES1A subfamily isozymes. The purified enzyme possessed temocapril hydrolase activity, and it was found to contribute significantly to temocapril hydrolase activity in mouse liver microsomes. To identify the nucleotide sequences coding mouse CES1, antibody screening of a cDNA library was performed. The deduced amino acid sequence of the obtained cDNA, mCES1, exhibited striking similarity to those of CES1A isozymes. When expressed in Sf9 cells, recombinant mCES1 showed hydrolytic activity toward temocapril, as did purified mouse CES1. Based on these results, together with the findings that recombinant mouse CES1 had the same molecular weight of a subunit, the same isoelectronic point, and the same native protein mass as those of purified mouse CES1, it was concluded that mCES1 encoded mouse CES1. Furthermore, tissue expression profiles of mCES1 were found to be very similar to those of the human CES1 isozyme. This finding, together with our other results, suggests that mCES1 shares many biological properties with the human CES1 isozyme. The present study has provided useful information for study of metabolism and disposition of ester-prodrugs as well as ester-drugs.

Hydrolysis of triglycerides is central to energy homeostasis in white adipose tissue (WAT). Hormone-sensitive lipase (HSL) was previously felt to mediate all lipolysis in WAT. Surprisingly, HSL-deficient mice show active HSL-independent lipolysis, suggesting that other lipase(s) also mediate triglyceride hydrolysis. To clarify this, we used functional proteomics to detect non-HSL lipase(s) in mouse WAT. After cell fractionation of intraabdominal WAT, most non-HSL neutral lipase activity is localized in the 100,000 x g infranatant and fat cake fractions. By oleic acid-linked agarose chromatography of infranatant followed by elution in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid gradient, we identified two peaks of esterase activity using p-nitrophenyl butyrate as a substrate. One of the peaks contained most of the lipase activity. In the corresponding fractions, gel permeation chromatography and SDS-PAGE, followed by tandem mass spectrometric analysis of excised Coomassie Blue-stained peptides, revealed carboxylesterase 3 (triacylglycerol hydrolase (TGH); EC 3.1.1.1). TGH is also the principle lipase of WAT fat cake extracts. Partially purified WAT TGH had lipase activity as well as lesser but detectable neutral cholesteryl ester hydrolase activity. Western blotting of subcellular fractions of WAT and confocal microscopy of fibroblasts following in vitro adipocytic differentiation are consistent with a distribution of TGH to endoplasmic reticulum, cytosol, and the lipid droplet. TGH is responsible for a major part of non-HSL lipase activity in WAT in vitro and may mediate some or all HSL-independent lipolysis in adipocytes.

Only a small proportion of the mouse genome is transcribed into mature messenger RNA transcripts. There is an international collaborative effort to identify all full-length mRNA transcripts from the mouse, and to ensure that each is represented in a physical collection of clones. Here we report the manual annotation of 60,770 full-length mouse complementary DNA sequences. These are clustered into 33,409 'transcriptional units', contributing 90.1% of a newly established mouse transcriptome database. Of these transcriptional units, 4,258 are new protein-coding and 11,665 are new non-coding messages, indicating that non-coding RNA is a major component of the transcriptome. 41% of all transcriptional units showed evidence of alternative splicing. In protein-coding transcripts, 79% of splice variations altered the protein product. Whole-transcriptome analyses resulted in the identification of 2,431 sense-antisense pairs. The present work, completely supported by physical clones, provides the most comprehensive survey of a mammalian transcriptome so far, and is a valuable resource for functional genomics.

A novel murine cDNA for triacylglycerol hydrolase (TGH), an enzyme that is involved in mobilization of triacylglycerol from storage pools in hepatocytes, has been cloned and expressed. The cDNA consists of 1962 bp with an open reading frame of 1695 bp that encodes a protein of 565 amino acids. Murine TGH is a member of the CES1A class of carboxylesterases and shows a significant degree of identity to other carboxylesterases from rat, monkey and human. Expression of the cDNA in McArdle RH7777 hepatoma cells showed a 3-fold increase in the hydrolysis of p-nitrophenyl laurate compared to vector-transfected cells. The highest expression of TGH was observed in the livers of mice, with lower expression in kidney, heart, adipose and intestinal (duodenum/jejunum) tissues. The murine gene that encodes TGH was cloned and exon-intron boundaries were determined. The gene spans approx. 35 kb and contains 14 exons. The results will permit future studies on the function of this gene via gene-targeting experiments and analysis of transcriptional regulation of the TGH gene.

The DNA sequence encoding a novel human brain carboxylesterase (CES) has been determined. The protein is predicted to have 567 amino acids, including conserved motifs, such as GESAGG, GXXXXEFG, and GDHGD which comprise a catalytic triad, and the endoplasmic reticulum retention motif (HXEL-COOH) observed in CES families. Their gene products exhibited hydrolase activity towards temocapril, p-nitrophenyl-acetate and long-chain acyl-CoA. Since the molecular masses of these gene products are similar to those that exist in capillary endothelial cells of the human brain [Yamamda et al. (1994) Brain Res. 658, 163-167], these CES isozymes may function as a blood-brain barrier to protect the central nervous system from ester or amide compounds.