Methionine transsulfuration is increased during sepsis in rats.

Methionine transsulfuration in plasma and liver, and plasma methionine and cysteine kinetics were investigated in vivo during the acute phase of sepsis in rats. Rats were infected with an intravenous injection of live Escherichia coli, and control pair-fed rats were injected with saline. Two days after injection, the rats were infused for 6 h with [(35)S]methionine and [(15)N]cysteine. Transsulfuration was measured from the transfer rate of (35)S from methionine to cysteine. Liver cystathionase activity was also measured. Infection significantly increased (P < 0.05) the contribution of transsulfuration to cysteine flux in both plasma and liver (by 80%) and the contribution of transsulfuration to plasma methionine flux (by 133%). Transsulfuration measured in plasma was significantly (P < 0.05) higher in infected rats than in pair-fed rats (0.68 and 0.25 micromol. h(-1). 100 g(-1), respectively). However, liver cystathionase specific activity was decreased by 17% by infection (P < 0.05). Infection increased methionine flux (16%, P < 0.05) less than cysteine flux (38%, P < 0.05). Therefore, the plasma cysteine flux was higher than that predicted from estimates of protein turnover based on methionine data, probably because of enhanced glutathione turnover. Taken together, these results suggest an increased cysteine requirement in infection.

Glutathione turnover is increased during the acute phase of sepsis in rats.

Glutathione metabolism during infection has been poorly documented. Glutathione concentrations and synthesis rates were studied in infected rats (2 d after infection) and in pair-fed controls. Glutathione synthesis rates were determined in liver, spleen, lung, small and large intestine, skeletal muscle, heart and blood by a 4-h or 6-h (15)N cysteine infusion. The activities of four hepatic enzymes involved in glutathione metabolism were also determined. Glutathione synthesis rates were significantly greater in liver (+465%), spleen (+388%), large intestine (+109%), lung (+100%), muscle (+91%) and heart (+80%) of infected rats compared with pair-fed controls. Glutathione concentrations were also greater in these tissues but were unaffected in small intestine and lower in blood. In keeping with the stimulation of liver glutathione synthesis, the activities of liver gamma-glutamyl-cysteine synthetase and glutathione reductase were significantly greater in liver of infected rats than of pair-fed rats. From the present study, we estimate that glutathione synthesis accounts for at least 40% of the enhanced cysteine utilization during infection. This increased utilization may be the primary cause of an enhanced cysteine requirement in infection.

Gas chromatographic-mass spectrometric analysis of stable isotopes of cysteine and glutathione in biological samples.

A gas chromatographic-mass spectrometric (GC-MS) procedure for the determination of stable isotope labelled glutathione has been applied to animal and human samples. The method, based on preparation of the N,S-ethoxycarbonyl methyl ester derivative of the intact peptide, is rapid and requires little or minor tissue treatment. The same method was applied to cysteine. The method was found to be reliable in terms of within-day and between-day precision, accuracy and linearity. The procedure was applied in humans and animals to determine in vivo the glutathione fractional synthesis rate using labelled cysteine infusion. The glutathione fractional synthesis rate was found to be 22.5%/day in blood from a healthy volunteer and 337+/-29%/day in rat liver.

Lower recovery of muscle protein lost during starvation in old rats despite a stimulation of protein synthesis.

Sarcopenia could result from the inability of an older individual to recover muscle lost during catabolic periods. To test this hypothesis, we compared the capacity of 5-day-refed 12- and 24-mo-old rats to recover muscle mass lost after 10 days without food. We measured gastrocnemius and liver protein synthesis with the flooding-dose method and also measured nitrogen balance, 3-methylhistidine excretion, and the gene expression of components of proteolytic pathways in muscle comparing fed, starved, and refed rats at each age. We show that 24-mo-old rats had an altered capacity to recover muscle proteins. Muscle protein synthesis, inhibited during starvation, returned to control values during refeeding in both age groups. The lower recovery in 24-mo-old rats was related to a lack of inhibition of muscle proteolysis during refeeding. The level of gene expression of components of the proteolytic pathways did not account for the variations in muscle proteolysis at both ages. In conclusion, this study highlights the role of muscle proteolysis in the lower recovery of muscle protein mass lost during catabolic periods.

Metabolism of cysteine is modified during the acute phase of sepsis in rats.

In vivo cysteine metabolism during the inflammatory state has been studied minimally. We investigated cysteine metabolism (i.e. taurine, sulfate and glutathione formation) using a single dose of [35S] cysteine in septic rats that had been injected with live Escherichia coli into the tail vein and in control, pair-fed rats. Cysteine metabolites were separated by ion exchange chromatography, and radioactivity was counted in the different fractions. Radioactivity incorporated in tissue proteins was also measured after protein precipitation. [35S]Sulfate production was significantly lower in septic rats than in pair-fed rats. [35S]Taurine contents were significantly lower only in kidneys, spleen and gastrointestinal tract of septic rats. The higher production of [35S] taurine in the livers (the major site of taurine production) of septic rats could have a protective effect against oxidation. Glutathione concentrations were also significantly greater in liver, spleen, kidneys and gastrocnemius muscle of septic rats, presumably in order to combat oxidative stress induced by sepsis. [35S]Cysteine incorporation in glutathione was significantly higher in spleen and kidneys but not in liver of septic rats compared to pair-fed rats. This could be explained by the fact that, in liver, a greater amount of labeled glutathione had been utilized for host defense, or by a high level in glutathione turnover. Finally, [35S]cysteine incorporation into protein, in septic rats, was significantly greater than in pair-fed rats in spleen, lung and particulary in whole plasma proteins other than albumin, which mainly represent the acute-phase proteins. These data suggest an increased requirement for cysteine during sepsis in rats.

Muscle and liver protein synthesis adapt efficiently to food deprivation and refeeding in 12-month-old rats.

Our aim was to analyze mechanisms involved in the adaptation of protein metabolism to food deprivation and refeeding in adult rats. Twelve-month-old rats, which had been food-deprived for 113 h and refed for 6 h, were injected subcutaneously with a flooding dose of valine (with 50% [1-13C]-L-valine) to measure in vivo protein synthesis in tibialis anterior, soleus and liver. Protein and RNA contents were also measured. In both muscles, protein mass was maintained during food deprivation. Due to a drop in protein synthetic capacity (Cs), total and myofibrillar protein synthesis rates were reduced in food-deprived rats and were not stimulated by a 6-h refeeding. In contrast, protein levels were maintained lower than RNA levels in liver during food deprivation, and Cs was higher than in fed rats. Protein synthesis rates and ribosomal efficiency were reduced in food-deprived rats. Due to maintenance of protein synthetic capacity, there was a rapid stimulation of liver protein synthesis with refeeding, which induced a significant rise in protein mass (also related to an inhibition of protein degradation). In conclusion, coordinated responses of liver and muscles allowed a sparing of muscle proteins during food deprivation and a rapid recovery of liver proteins during refeeding. Control of ribosome quantity could play a critical role in these adaptations in tissue protein synthesis in adult rats.