Down-regulation of Hepatic Urea Synthesis by Oxypurines

Abstract

We previously reported that isobutylmethylxanthine (IBMX), a derivative of oxypurine, inhibits citrulline synthesis by an as yet unknown mechanism. Here, we demonstrate that IBMX and other oxypurines containing a 2,6-dione group interfere with the binding of glutamate to the active site of N-acetylglutamate synthetase (NAGS), thereby decreasing synthesis of N-acetylglutamate, the obligatory activator of carbamoyl phosphate synthase-1 (CPS1). The result is reduction of citrulline and urea synthesis. Experiments were performed with 15 N-labeled substrates, purified hepatic CPS1, and recombinant mouse NAGS as well as isolated mitochondria. We also used isolated hepatocytes to examine the action of various oxypurines on ureagenesis and to assess the ameliorating affect of N-carbamylglutamate and/or L-arginine on NAGS inhibition. Among various oxypurines tested, only IBMX, xanthine, or uric acid significantly increased the apparent K m for glutamate and decreased velocity of NAGS, with little effect on CPS1. The inhibition of NAGS is time-and dose-dependent and leads to decreased formation of the CPS1-N-acetylglutamate complex and consequent inhibition of citrulline and urea synthesis. However, such inhibition was reversed by supplementation with N-carbamylglutamate. The data demonstrate that xanthine and uric acid, both physiologically occurring oxypurines, inhibit the hepatic synthesis of N-acetylglutamate. An important and novel concept emerging from this study is that xanthine and/or uric acid may have a role in the regulation of ureagenesis and, thus, nitrogen homeostasis in normal and disease states.

Effects of a glucokinase activator on hepatic intermediary metabolism: study with 13C-isotopomer-based metabolomics

“…In addition, the results demonstrate that the production of 13 C-labelled glutamate through the TCA cycle was significantly increased (Figure 7). Together, increased mitochondrial levels of glutamate and acetyl-CoA, the precursors of NAG [12], led to increased NAG synthesis, activation of carbamoylphosphate synthetase-I and greater mitochondrial citrulline synthesis [12,37,38]. The results of the present study are consistent with the concept that the synthesis of citrulline and urea is determined in large measure by NAG synthesis, with which the rate of ureagenesis is linearly correlated.…”

“…This increase is mainly due to a significantly augmented ( P = 0.028) synthesis of 13 C-labelled NAG. Because NAG is synthesized from acetyl-CoA and glutamate [12], the 13 C mass isotopomers profile for NAG indicates that [ 13 C]NAG was generated from [ 13 C 2 ]acetyl-CoA and the various 13 C mass isotopomers of glutamate, as depicted in Figure 7(A). Thus, consistent with the augmented production of 13 C-labelled metabolites by GKA (Figures 5–7), the increased production of 13 C-labelled NAG was probably mediated by increased availability of [ 13 C]pyruvate and its metabolism to acetyl-CoA.…”

“…Hepatocytes were prepared from liver of fed rats by a two-step procedure as described previously [11,12]. Briefly, after washing the liver with oxygenated Krebs buffer (pH 7.5), isolated liver was perfused for 10–15 min with 0.03% collagenase in Krebs buffer (37 °C, pH 7.5).…”

Renal Oxidative Stress Induced by Long-Term Hyperuricemia Alters Mitochondrial Function and Maintains Systemic Hypertension

“…Interestingly Tempol also prevented mitochondrial uric acid accumulation. Mitochondrial uric acid results from at least three mechanisms: mitochondrial xanthine oxidase activity [ 22 ], mitochondrial purine catabolism [ 23 ], and uptake from cytoplasmic space [ 24 ]. Regulation of systemic and intracellular uric acid levels is a complex process that requires the participation of several different transporters for its uptake and its efflux.…”

A role for bacterial urease in gut dysbiosis and Crohn’s disease

“…All groups were gavaged with 50 mg of [ 15 N 2 ]urea per mouse daily for 7 days. 15 N enrichment in amino acids was determined by liquid/gas chromatography–MS and HPLC (49, 50). …”