Is N-Carbamoyl Putrescine, the Decarboxylation Derivative of Citrulline, a Regulator of Muscle Protein Metabolism in Rats?

Abstract

N-carbamoyl putrescine (NCP), the decarboxylation derivative of citrulline, metabolically related to polyamines, may exert biological effects in mammals. The aim of this study was (i) to evaluate the nutritional properties of NCP in healthy rats and (ii) to determine the effect of NCP administration on muscle metabolism in malnourished old rats. The nutritional properties of NCP were first evaluated in 20 8-week-old male rats randomized to receive for two weeks a standard diet either alone (C group) or supplemented with NCP, 5 or 50 mg/kg/d. In a second study, 29 malnourished 18-month-old male rats were studied either before or after a 4-day refeeding with a standard diet either alone (REN group) or supplemented with NCP, 1 or 10 mg/kg/d. NCP had no effect on weight gain and body composition in either of the two studies. In healthy rats, muscle protein content was significantly increased in the soleus with NCP 5 mg/kg/d. A decrease in plasma glutamine and kidney spermine was observed at the 50 mg/kg/d dose; otherwise, no significant changes in plasma chemistry and tissue polyamines were observed. In malnutrition-induced sarcopenic old rats, refeeding with NCP 10 mg/kg/d was associated with higher tibialis weight and a trend for increased protein content in extensor digitorum longus (EDL). While the muscle protein synthesis rate was similar between groups, ribosomal protein S6 kinase was increased in tibialis and higher in the EDL in NCP-treated rats. The muscle RING-finger protein-1 expression was decreased in tibialis and urinary 3-methyl-histidine to creatinine ratio slightly lower with the supply of NCP. However, this initial period of refeeding was also associated with elevated fasted plasma triglycerides and glucose, significant in NCP groups, suggesting glucose intolerance and possibly insulin resistance. NCP was well-tolerated in healthy young-adults and in malnourished old rats. In healthy adults, NCP at 5 mg/kg/d induced a significant increase in protein content in the soleus, a type I fiber-rich muscle. In malnourished old rats, NCP supply during refeeding, may help to preserve lean mass by limiting protein breakdown; however, these effects may be limited in our model by a possible immediate refeeding-associated glucose intolerance.

Keywords: N-carbamoyl putrescine; catabolism; citrulline; malnutrition; muscle anabolism.

Figure 1

Citrulline, arginine and ornithine as precursors of potentially active amine derivatives. ADC: Arg decarboxylase; AS: argininosuccinate; AS: AS synthase; ASL: AS lyase; OAT: Orn aminotransferase; OCT: Orn carbamyl transferase; ODC: Orn decarboxylase; NOS: nitric oxide synthase; SpdS: spermidine synthase; SpmS: spermine synthase; ?: demonstrated in some vegetal cells and bacteria.

Figure 2

The effect of NCP on the mammalian muscle target of rapamycine complex 1 (mTORC1) signaling during refeeding of malnourished old rats. Malnutrition was induced in old rats by a dietary restriction to 50% of their spontaneous intakes for 6 weeks. Thereafter, eight rats (n = 8, DEN group) were sacrificed immediately, while the animals of the other groups received, for 4 days, their standard diet at 100% of their spontaneous food intake alone (n = 9, REN group) or with NCP, 1 mg/kg/d (n = 9, NCP1 group) or 10 mg/kg/d (n = 8, NCP10 group). Phosphorylation status of p70 ribosomal protein S6 kinase 1 (S6K) (A,B,C) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) (D,E,F) were measured at the end of this 4-day period in fasted animals by western blot in the extensor digitaris longus (A,D), soleus (B,E) and tibialis (C,F). All values (means ± SEMs) are expressed as the ratios of each phosphorylated protein to their total expression. One-way ANOVA and post-hoc Tukey–Kramer; statistical significance: p < 0.05. * p < 0.05 versus DEN.

Figure 3

The effect of NCP on expression of Murf1 during refeeding of malnourished old rats. Malnutrition was induced in old rats by a dietary restriction to 50% of their spontaneous intakes for 6 weeks. Thereafter, eight rats (n = 8, DEN group) were sacrificed immediately while the animals of the other groups received, for 4 days, their standard diet at 100% of their spontaneous food intake alone (n = 9, REN group) or with NCP, 1 mg/kg/d (n = 9, NCP1 group) or 10 mg/kg/d (n = 8, NCP10 group). Expression of Murf1 was measured at the end of this 4-day period, in fasted animals, in their extensor digitaris longus (A), soleus (B) and tibialis (C) by western blot. All values (means ± SEMs) are expressed as ratios to a reference gene (GAPDH). One-way ANOVA and post-hoc Tukey–Kramer or Kruskal–Wallis and post-hoc Dunn test; statistical significance: p < 0.05. * p < 0.05 versus DEN group. $ p < 0.05 versus REN group.