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

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(Delta 19/Delta 19), 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/EBP alpha and C/EBP beta. 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(Delta 19/Delta 19), 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.