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Ammonia-lowering activities and carbamoyl phosphate synthetase 1 (Cps1) induction mechanism of a natural flavonoid.

Nohara K, Shin Y, Park N, Jeong K, He B, Koike N, Yoo SH, Chen Z - Nutr Metab (Lond) (2015)

Bottom Line: Expression of other urea cycle genes was also decreased by HFD relative to RC and again restored by NOB to varying degrees, which, in conjunction with Cps1 promoter reporter analysis, suggested a C/EBP-dependent mechanism for the co-induction of urea cycle genes by NOB.In comparison, HPD markedly increased CPS1 levels relative to RC, yet NOB did not further enrich CPS1 to a significant extent.NOB, a dietary flavonoid, exhibits a broad activity in ammonia control across varying diets, and regulates urea cycle function via C/EBP-and clock-dependent regulatory mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, 6431 Fannin Street, MSB 6.200, Houston, TX 77030 USA.

ABSTRACT

Objective: Ammonia detoxification is essential for physiological well-being, and the urea cycle in liver plays a predominant role in ammonia disposal. Nobiletin (NOB), a natural dietary flavonoid, is known to exhibit various physiological efficacies. In the current study, we investigated a potential role of NOB in ammonia control and the underlying cellular mechanism.

Materials/methods: C57BL/6 mice were fed with regular chow (RC), high-fat (HFD) or high-protein diet (HPD) and treated with either vehicle or NOB. Serum and/or urine levels of ammonia and urea were measured. Liver expression of genes encoding urea cycle enzymes and C/EBP transcription factors was determined over the circadian cycle. Luciferase reporter assays were carried out to investigate function of CCAAT consensus elements on the carbamoyl phosphate synthetase (Cps1) gene promoter. A circadian clock-deficient mouse mutant, Clock (Δ19/Δ19) , was utilized to examine a requisite role of the circadian clock in mediating NOB induction of Cps1.

Results: NOB was able to lower serum ammonia levels in mice fed with RC, HFD or HPD. Compared with RC, HFD repressed the mRNA and protein expression of Cps1, encoding the rate-limiting enzyme of the urea cycle. Interestingly, NOB rescued CPS1 protein levels under the HFD condition via induction of the transcription factors C/EBPα and C/EBPβ. Expression of other urea cycle genes was also decreased by HFD relative to RC and again restored by NOB to varying degrees, which, in conjunction with Cps1 promoter reporter analysis, suggested a C/EBP-dependent mechanism for the co-induction of urea cycle genes by NOB. In comparison, HPD markedly increased CPS1 levels relative to RC, yet NOB did not further enrich CPS1 to a significant extent. Using the circadian mouse mutant Clock (Δ19/Δ19) , we also showed that a functional circadian clock, known to modulate C/EBP and CPS1 expression, was required for NOB induction of CPS1 under the HFD condition.

Conclusion: NOB, a dietary flavonoid, exhibits a broad activity in ammonia control across varying diets, and regulates urea cycle function via C/EBP-and clock-dependent regulatory mechanisms.

No MeSH data available.


Related in: MedlinePlus

C/EBP binding site mutations in proximal and distal Cps1 enhancer regions impaired NOB-mediated reporter activation. a Diagram of distal and proximal C/EBP sites on the Cps1 promoter region. b and c Hepa1-6 cells were transfected with reporter constructs containing wild-type (WT; white bars) and C/EBP binding site mutated (black bars) enhancer regions, and treated with NOB at the indicated doses. Co-transfection of C/EBPα expression construct was also carried out in parallel. Each value represented mean ± SEM of three replicates from a single assay. The results are representative of at least three independent experiments. b and c show results from reporter constructs containing distal and proximal WT and mutant enhancers respectively. Two-way ANOVA with Bonferroni post-hoc tests: significant main effects of plasmid construct, (b) F = 81.43, p < 0.001, (c) F = 254.97, p < 0.0001; NOB concentration effect, (b) F = 47.39, p < 0.0001, (c) F = 42.01, p < 0.0001; and a significant interaction between constructs and NOB concentration, (b) F = 23, p < 0.0001, (c) F = 24.37, p < 0.0001
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Fig4: C/EBP binding site mutations in proximal and distal Cps1 enhancer regions impaired NOB-mediated reporter activation. a Diagram of distal and proximal C/EBP sites on the Cps1 promoter region. b and c Hepa1-6 cells were transfected with reporter constructs containing wild-type (WT; white bars) and C/EBP binding site mutated (black bars) enhancer regions, and treated with NOB at the indicated doses. Co-transfection of C/EBPα expression construct was also carried out in parallel. Each value represented mean ± SEM of three replicates from a single assay. The results are representative of at least three independent experiments. b and c show results from reporter constructs containing distal and proximal WT and mutant enhancers respectively. Two-way ANOVA with Bonferroni post-hoc tests: significant main effects of plasmid construct, (b) F = 81.43, p < 0.001, (c) F = 254.97, p < 0.0001; NOB concentration effect, (b) F = 47.39, p < 0.0001, (c) F = 42.01, p < 0.0001; and a significant interaction between constructs and NOB concentration, (b) F = 23, p < 0.0001, (c) F = 24.37, p < 0.0001

Mentions: Previous studies have identified both proximal and distal C/EBP sites in the Cps1 promoter [16, 35, 53]. We next generated reporter constructs containing either the intact Cps1 promoter or mutant promoters respectively deficient in either C/EBP sites (Fig. 4a). NOB was found to dose-dependently activate reporter expression from the WT construct with an intact promoter (Fig. 4b and c). Distal mutations markedly repressed the reporter expression, yet seemed to at least partially retain NOB response as NOB increased mutant reporter expression relative to DMSO (Fig. 4b). In comparison, while not significantly affecting the baseline expression level, proximal mutations abolished NOB dose-dependent induction seen in the WT construct (Fig. 4c). C/EBPα co-transfection activated both proximal and distal WT reporter expression, but showed no effects on mutant constructs in the absence of NOB. These results suggested that the proximal C/EBP consensus site plays a major role in mediating the NOB induction of Cps1.Fig. 4


Ammonia-lowering activities and carbamoyl phosphate synthetase 1 (Cps1) induction mechanism of a natural flavonoid.

Nohara K, Shin Y, Park N, Jeong K, He B, Koike N, Yoo SH, Chen Z - Nutr Metab (Lond) (2015)

C/EBP binding site mutations in proximal and distal Cps1 enhancer regions impaired NOB-mediated reporter activation. a Diagram of distal and proximal C/EBP sites on the Cps1 promoter region. b and c Hepa1-6 cells were transfected with reporter constructs containing wild-type (WT; white bars) and C/EBP binding site mutated (black bars) enhancer regions, and treated with NOB at the indicated doses. Co-transfection of C/EBPα expression construct was also carried out in parallel. Each value represented mean ± SEM of three replicates from a single assay. The results are representative of at least three independent experiments. b and c show results from reporter constructs containing distal and proximal WT and mutant enhancers respectively. Two-way ANOVA with Bonferroni post-hoc tests: significant main effects of plasmid construct, (b) F = 81.43, p < 0.001, (c) F = 254.97, p < 0.0001; NOB concentration effect, (b) F = 47.39, p < 0.0001, (c) F = 42.01, p < 0.0001; and a significant interaction between constructs and NOB concentration, (b) F = 23, p < 0.0001, (c) F = 24.37, p < 0.0001
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Fig4: C/EBP binding site mutations in proximal and distal Cps1 enhancer regions impaired NOB-mediated reporter activation. a Diagram of distal and proximal C/EBP sites on the Cps1 promoter region. b and c Hepa1-6 cells were transfected with reporter constructs containing wild-type (WT; white bars) and C/EBP binding site mutated (black bars) enhancer regions, and treated with NOB at the indicated doses. Co-transfection of C/EBPα expression construct was also carried out in parallel. Each value represented mean ± SEM of three replicates from a single assay. The results are representative of at least three independent experiments. b and c show results from reporter constructs containing distal and proximal WT and mutant enhancers respectively. Two-way ANOVA with Bonferroni post-hoc tests: significant main effects of plasmid construct, (b) F = 81.43, p < 0.001, (c) F = 254.97, p < 0.0001; NOB concentration effect, (b) F = 47.39, p < 0.0001, (c) F = 42.01, p < 0.0001; and a significant interaction between constructs and NOB concentration, (b) F = 23, p < 0.0001, (c) F = 24.37, p < 0.0001
Mentions: Previous studies have identified both proximal and distal C/EBP sites in the Cps1 promoter [16, 35, 53]. We next generated reporter constructs containing either the intact Cps1 promoter or mutant promoters respectively deficient in either C/EBP sites (Fig. 4a). NOB was found to dose-dependently activate reporter expression from the WT construct with an intact promoter (Fig. 4b and c). Distal mutations markedly repressed the reporter expression, yet seemed to at least partially retain NOB response as NOB increased mutant reporter expression relative to DMSO (Fig. 4b). In comparison, while not significantly affecting the baseline expression level, proximal mutations abolished NOB dose-dependent induction seen in the WT construct (Fig. 4c). C/EBPα co-transfection activated both proximal and distal WT reporter expression, but showed no effects on mutant constructs in the absence of NOB. These results suggested that the proximal C/EBP consensus site plays a major role in mediating the NOB induction of Cps1.Fig. 4

Bottom Line: Expression of other urea cycle genes was also decreased by HFD relative to RC and again restored by NOB to varying degrees, which, in conjunction with Cps1 promoter reporter analysis, suggested a C/EBP-dependent mechanism for the co-induction of urea cycle genes by NOB.In comparison, HPD markedly increased CPS1 levels relative to RC, yet NOB did not further enrich CPS1 to a significant extent.NOB, a dietary flavonoid, exhibits a broad activity in ammonia control across varying diets, and regulates urea cycle function via C/EBP-and clock-dependent regulatory mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston, 6431 Fannin Street, MSB 6.200, Houston, TX 77030 USA.

ABSTRACT

Objective: Ammonia detoxification is essential for physiological well-being, and the urea cycle in liver plays a predominant role in ammonia disposal. Nobiletin (NOB), a natural dietary flavonoid, is known to exhibit various physiological efficacies. In the current study, we investigated a potential role of NOB in ammonia control and the underlying cellular mechanism.

Materials/methods: C57BL/6 mice were fed with regular chow (RC), high-fat (HFD) or high-protein diet (HPD) and treated with either vehicle or NOB. Serum and/or urine levels of ammonia and urea were measured. Liver expression of genes encoding urea cycle enzymes and C/EBP transcription factors was determined over the circadian cycle. Luciferase reporter assays were carried out to investigate function of CCAAT consensus elements on the carbamoyl phosphate synthetase (Cps1) gene promoter. A circadian clock-deficient mouse mutant, Clock (Δ19/Δ19) , was utilized to examine a requisite role of the circadian clock in mediating NOB induction of Cps1.

Results: NOB was able to lower serum ammonia levels in mice fed with RC, HFD or HPD. Compared with RC, HFD repressed the mRNA and protein expression of Cps1, encoding the rate-limiting enzyme of the urea cycle. Interestingly, NOB rescued CPS1 protein levels under the HFD condition via induction of the transcription factors C/EBPα and C/EBPβ. Expression of other urea cycle genes was also decreased by HFD relative to RC and again restored by NOB to varying degrees, which, in conjunction with Cps1 promoter reporter analysis, suggested a C/EBP-dependent mechanism for the co-induction of urea cycle genes by NOB. In comparison, HPD markedly increased CPS1 levels relative to RC, yet NOB did not further enrich CPS1 to a significant extent. Using the circadian mouse mutant Clock (Δ19/Δ19) , we also showed that a functional circadian clock, known to modulate C/EBP and CPS1 expression, was required for NOB induction of CPS1 under the HFD condition.

Conclusion: NOB, a dietary flavonoid, exhibits a broad activity in ammonia control across varying diets, and regulates urea cycle function via C/EBP-and clock-dependent regulatory mechanisms.

No MeSH data available.


Related in: MedlinePlus