Figure 1.
Hepatic CES1 is regulated by nutritional status.
(A–C) Hepatic mRNA levels in ob/ob (A) and db/db mice (B) mice were determined by qRT-PCR and protein levels determined by Western blot assays (C, top) (n = 4–6 mice per group). In (C, bottom), protein levels were also quantified by Image J. (D) C57BL/6 mice were treated with either vehicle (0.1 M sodium citrate, pH 4.5) or streptozotocin (STZ) (50 mg/kg/d) for 5 days. Seven days after STZ treatment, mice were euthanized and hepatic mRNA levels were quantified (n = 5 mice per group). (E) Wild-type mice were fed a chow or high fat/high cholesterol (HFHC) diet (21% fat, 1.5% cholesterol) for 3 weeks and hepatic mRNA levels were determined (n = 8 mice per group). (F) C57BL/6 mice were fed a chow diet, or fasted for 3, 8, 24 h, or fasted for 24 h followed by refed for 24 h (n = 5 mice per group). Hepatic protein levels were determined by Western blot assays (top) and then quantified (bottom). Pgc-1α, peroxisome proliferator-activated receptor gamma coactivator-1α. Abca1, ATP-binding cassette (ABC) transporter A1. Abcg5, ABC transporter G5. Pepck, phosphoenolpyruvate carboxykinase. *p<0.05, **p<0.01.
Figure 2.
Hepatic CES1 is regulated by glucose but not insulin.
(A–C) C57BL/6 mice were fasted for 16 h, followed by gavage with saline or glucose (8 g/kg) (n = 6 mice per group). Three hours after second oral gavage, hepatic mRNA levels (A) and protein levels (B) were determined. Hepatic CES1 protein levels were quantified (C). L-PK serves as a positive control in (A). (D) C57BL/6 mice were fasted for 16 h, followed by i.p. injection of either saline or insulin (0.8 units/kg) (n = 5 mice per group). After 3 h, mice were euthanized. Srebp-1c serves as a positive control. (E) Mouse primary hepatocytes were isolated and cultured in dulbecco's modified eagle medium (DMEM) plus 10% fetal bovine serum (FBS) overnight, followed by serum-free fasting for 8 h. Cells were then treated with either normal (5.5 mM) or high (27.5 mM) glucose for additional 24 h prior to quantification of mRNA levels. Fas serves as a positive control. (F) CES1 promoter-luciferase constructs were transfected into HepG2 cells, then treated with 5.5 mM or 27.5 mM glucose. After 36 h, luciferase activity was determined. Srebp-1c, sterol response element binding protein-1c. L-PK, liver type pyruvate kinase. Fas, fatty acid synthase. RLU, relative luciferase units. *p<0.05, **p<0.01.
Figure 3.
ACL is required for glucose-induced hepatic CES1 expression.
(A) C57BL/6 mice were injected i.v. with adenovirus expressing GFP or ChREBP. After 5 days, hepatic mRNA levels were determined by qPCR (n = 7 mice per group). (B–F) C57BL/6 mice were injected with adenovirus expressing shLacZ or shAcl (n = 6 mice per group). After 5 days, mice were gavaged with either saline or glucose (8 g/kg). Hepatic mRNA levels of Acl (B), Ces1 (C) and L-pk (F) were determined. Hepatic protein levels were determined by Western blot assays (D) and CES1 protein levels quantified (E). *p<0.05, **p<0.01.
Figure 4.
ACL is required for glucose-mediated acetylation of histones (H3, H4) in the CES1 chromatin.
(A, B) C57BL/6 mice were treated as described in Fig. 3B–F. Liver lysates were used for ChIP assay to determine acetylation of histone 3 (AcH3) (A) and histone 4 (AcH4) (B). *p<0.05, **p<0.01.
Figure 5.
CES1 regulates postprandial levels.
(A–H) C57BL/6 mice were injected with Ad-shLacZ or Ad-shCes1. After 5 days, mice were fasted for 16 h followed by gavage with saline or glucose (8 g/kg) (n = 6 mice per group). Blood glucose levels were measured 1 h after gavage using a glucometer (A). Mice were then sacrificed 3 hours after gavage. Hepatic triglyceride (TG) (B) and free fatty acid (FFA) (C) levels were analyzed. Hepatic protein levels were assessed by Western blot assays (D) and then the ratio of p-AKT to total AKT was quantified (E). Hepatic mRNA levels of Ces1 (F), PEPCK (G) and G6Pase (H) were determined by qRT-PCR. (I) Reciprocal regulation between plasma glucose and hepatic CES1. Elevated plasma glucose induces hepatic CES1, which in turn helps lower plasma glucose levels likely via increasing peripheral insulin sensitivity. AKT, protein kinase B. *p<0.05 **p<0.01.