Creatine synthesis: hepatic metabolism of guanidinoacetate and creatine in the rat in vitro and in vivo

Abstract

Since creatinine excretion reflects a continuous loss of creatine and creatine phosphate, there is a need for creatine replacement, from the diet and/or by de novo synthesis. Creatine synthesis requires three amino acids, methionine, glycine, and arginine, and two enzymes, l-arginine:glycine amidinotransferase (AGAT), which produces guanidinoacetate acid (GAA), and guanidinoacetate methyltransferase (GAMT), which methylates GAA to produce creatine. In the rat, high activities of AGAT are found in the kidney, whereas high activities of GAMT occur in the liver. Rat hepatocytes readily convert GAA to creatine; this synthesis is stimulated by the addition of methionine, which increases cellular S-adenosylmethionine concentrations. These same hepatocytes are unable to produce creatine from methionine, arginine, and glycine. (15)N from (15)NH(4)Cl is readily incorporated into urea but not into creatine. Hepatic uptake of GAA is evident in vivo by livers of rats fed a creatine-free diet but not when rats were fed a creatine-supplemented diet. Rats fed the creatine-supplemented diet had greatly decreased renal AGAT activity and greatly decreased plasma [GAA] but no decrease in hepatic GAMT or in the capacity of hepatocytes to produce creatine from GAA. These studies indicate that hepatocytes are incapable of the entire synthesis of creatine but are capable of producing it from GAA. They also illustrate the interplay between the dietary provision of creatine and its de novo synthesis and point to the crucial role of renal AGAT expression in regulating creatine synthesis in the rat.

Fig. 1.

Creatine biosynthetic pathway. HA, h′epatic artery; PV, portal vein; HV, hepatic vein; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; GAMT, guanidinoacetate N-methyltransferase; AGAT, L-arginine:glycine amidinotransferase; GAA, guanidinoacetate acid.

Fig. 2.

Effect of [GAA] on creatine synthesis. Hepatocytes were incubated with varying [GAA], with (▴) or without (▪) 0.5 mM methionine. Each flask contained an average of 6.5 mg dry mass of cells and was incubated at 37°C for 60 min (n = 4). Data are expressed as means ± SD and fit to a curve using nonlinear regression.

Fig. 3.

Differences in plasma GAA concentration across the liver of rats fed a diet supplemented with 0.4% creatine (A) and rats fed a diet free of creatine (B). Plasma values are represented as follows: HA (white), PV (light gray), HV (dark gray), and concentration difference across liver (hatched). The different letters (a and b) indicate a statistically significant difference (P < 0.05) between the different blood vessels. *Significant difference (P < 0.05) between the vascular inflow and outflow values. The hepatic inflow was calculated as described in Hepatic blood sampling. Data are expressed as means ± SD (n = 6).

Fig. 4.

Differences in plasma creatine concentration across the liver of rats fed a diet supplemented with 0.4% creatine (A) and rats fed a diet free of creatine (B). Plasma values are represented as follows: arterial (Art; white), PV (light gray), HV (dark gray), and concentration difference across liver (hatched). ^aThere was no significant difference (P > 0.05) between the creatine concentrations in the different vessels. The data are expressed as means ± SD (n = 6).

Fig. 5.

Enzyme activities of AGAT and GAMT and creatine production by hepatocytes isolated from rats fed different diets. A: renal AGAT activity. B: hepatic GAMT activity. C: creatine synthesis in hepatocytes. White bars, rats fed the creatine-free diets; dark gray bars, rats fed the creatine-supplemented diets. Hepatocytes were incubated with 0.5 mM methionine and 125 μM GAA at 37°C for 60 min. All values are expressed as means ± SD. *Statistical significance P < 0.05 (n = 4).