Carboxylesterase 1 family knockout alters drug disposition and lipid metabolism

Abstract

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.

Keywords: Adipose tissue; Capecitabine; Ces1/CES1 enzymes; Inflammation; Irinotecan; Lipid homeostasis; Mouse models; Triglyceride mobilization.

Graphical abstract

Figure 1

Generation of Ces1^–/– mice. (A) Schematic overview of the strategy for deletion of the Ces1 cluster genes. The cutting sites (stippled lines) were targeted just upstream and downstream of the Ces1 cluster by CRISPR/Cas9 methodology. Subsequently the whole Ces1 cluster was deleted, yielding a product allele as depicted below the Ces1 locus (stippled line). Gene sizes and intergenic distances are not to scale. Each white box represents a single gene. (B) Detail of the Cas9/sgRNA/oligo targeting sites downstream and upstream of the Ces1 cluster. The sgRNA coding sequence is underlined and labeled in red. The PAM sequence is shown in blue. The Cas9 cutting sites are indicated by red arrows. In the oligo donor sequence, the loxP site is indicated in yellow, and the restriction site sequence in purple. The donor oligos contain 60 bp homologies on both sides flanking the planned double-strand breaks. (C) PCR analysis of all Ces1 genes in tail DNA of wild-type (WT) and Ces1^–/– mice; KO: Ces1 cluster knockout.

Figure 2

Ces1 loss strongly reduces irinotecan conversion to SN-38. Pharmacokinetics of irinotecan and SN-38 after oral or i.v. administration of irinotecan hydrochloride trihydrate (20 mg/kg) to male WT and Ces1^–/– mice. Plasma concentration versus time curves and AUCs of irinotecan and SN-38 after oral (A, B) or i.v. (C, D) administration of irinotecan. Tissue concentrations of irinotecan, SN-38 and SN-38-to-irinotecan ratios in liver (E, G) and kidney (F, H). Data are presented as mean ± SD (n = 4–5; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001 when compared with WT mice; the statistical calculation was performed after log-transformation of data; two-tailed unpaired Student’s t test).

Figure 3

Human CES1 expression in transgenic mice. (A) Schematic structure of the ApoE promoter-HCR1-driven expression cassette containing wild-type human CES1 cDNA. (B) Western blot analysis of crude membrane protein of liver, small intestine (SI) and kidney from WT, Ces1^–/– and TgCES1 mice. Crude membrane protein from human liver (HCM) was used as positive control. HCM: human crude liver membrane. (C) Immuno-histochemical staining of human CES1 in liver and small intestine of WT, Ces1^–/– and TgCES1 male mice. Scale bar: 300 μm.

Figure 4

Human CES1 transgenic mice exhibit enhanced metabolism of irinotecan to SN-38 in liver and kidney. Pharmacokinetics of irinotecan and SN-38 after i.v. administration of irinotecan hydrochloride trihydrate (20 mg/kg) to male WT, Ces1^–/– and TgCES1 mice. (A, B) Plasma SN-38-to-irinotecan ratio versus time curves and AUC ratios. Tissue concentrations of irinotecan, SN-38 and SN-38-to-irinotecan ratios in liver (C–E) and kidney (F–H). Data are presented as mean ± SD (n = 3–7; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001 when compared with WT mice; ⁺P < 0.05; ⁺⁺P < 0.01; ⁺⁺⁺P < 0.001; ⁺⁺⁺⁺P < 0.0001 when TgCES1 compared with Ces1^–/– mice; the statistical calculation was performed after log-transformation of data; one-way ANOVA followed by Tukey’s post hoc test).

Figure 5

Mouse Ces1 and human CES1 influence metabolite-to-capecitabine ratios in plasma. Pharmacokinetics of capecitabine and its 4 metabolites (5-DFCR, 5-DFUR, 5-FU and FBAL) after oral administration of capecitabine (500 mg/kg) to female WT, Ces1^–/– and TgCES1 mice. (A, B) Plasma 5-DFCR to capecitabine ratio versus time curves and AUC ratios. (C, D) Plasma 5-DFUR to capecitabine ratio versus time curves and AUC ratios. (E, F) Plasma 5-FU to capecitabine ratio versus time curves and AUC ratios. (G, H) Plasma FBAL to capecitabine ratio versus time curves and AUC ratios. Data are presented as mean ± SD (n = 4–7; ∗∗∗∗P < 0.0001 when compared with WT mice; ⁺P < 0.05; ⁺⁺P < 0.01 when TgCES1 compared with Ces1^–/– mice; the statistical calculation was performed after log-transformation of the data; one-way ANOVA followed by Tukey’s post hoc test).

Figure 6

Ces1 deficiency results in increased body weight, disrupted lipid homeostasis and hepatic expression of human CES1 reverses these phenotypes in male mice. Male WT, Ces1^–/– and TgCES1 mice on a medium-fat diet were analyzed. (A) Body weight-time curves between 6 and 15 weeks of age, with inset showing the area under the curves (n = 14–16). (B) Weight of different adipose tissue depots (n = 10–12, 12–13 weeks). (C) Haematoxylin and eosin staining of perigonadal white adipose tissue (top panels) and brown adipose tissue (bottom panels) (n = 10–12, 12–13 weeks). Scale bar: 200 μm. Data are presented as mean ± SD (∗P < 0.05; ∗∗P < 0.01 when Ces1^–/– compared with WT mice; ⁺P < 0.05; ⁺⁺P < 0.01; ⁺⁺⁺P < 0.001 when TgCES1 compared with Ces1^–/– mice; one-way ANOVA followed by Tukey’s post hoc test).

Figure 7

Hepatic expression of human CES1 increases triglyceride secretion from liver to plasma in male mice. (A) Plasma concentration of triglyceride (clinical chemistry analysis) of male mice (n = 7–21, 9–16 weeks). (B) Triglyceride secretion from liver to blood. Male mice (n = 7–9, 12–14 weeks) were i.p. injected with lipase inhibitor P-407, plasma was collected and measured at indicated time points. (C) Triglyceride concentration in liver (n = 10–12, 12–13 weeks). (D) Haematoxylin and eosin (top panels), Oil Red O (bottom panels) staining of liver (n = 8–12, 12–13 weeks). Scale bar: 200 μm. (E) RT-PCR analysis of fatty acid synthesis genes in liver (n = 6, 12–13 weeks). Data are presented as mean ± SD (∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001 when TgCES1 compared with WT mice; ⁺P < 0.05; ⁺⁺P < 0.01; ⁺⁺⁺P < 0.001; ⁺⁺⁺⁺P < 0.0001 when TgCES1 compared with Ces1^–/– mice; one-way ANOVA followed by Tukey’s post hoc test).

Figure 8

Glucose and insulin tolerance tests in male WT, Ces1^–/– and TgCES1 mice. (A, B) Male mice were fasted for 16 h, glucose (1 mg/g) was orally administered (n = 12, 11–12 weeks), and blood glucose was measured. (C, D) Male mice were fasted for 6 h, insulin (0.5 U/kg) was i.p. administered (n = 12, 11–12 weeks), and blood glucose was measured. Data are presented as mean ± SD (∗P < 0.05; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001 when Ces1^–/– compared with WT mice; ⁺P < 0.05; ⁺⁺P < 0.01; ⁺⁺⁺P < 0.001 when TgCES1 compared with Ces1^–/– mice; one-way ANOVA followed by Tukey’s post hoc test).

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