The availability of cysteine is thought to be the rate limiting factor for synthesis of the tripeptide glutathione (GSH), based on studies in rodents. GSH status is compromised in various disease states and by certain medications leading to increased morbidity and poor survival. To determine the possible importance of dietary cyst(e)ine availability for whole blood glutathione synthesis in humans, we developed a convenient mass spectrometric method for measurement of the isotopic enrichment of intact GSH and then applied it in a controlled metabolic study. Seven healthy male subjects received during two separate 10-day periods an L-amino acid based diet supplying an adequate amino acid intake or a sulfur amino acid (SAA) (methionine and cysteine) free mixture (SAA-free). On day 10, L-[1-13 C]cysteine was given as a primed, constant i.v. infusion (3mol⅐kg ؊1 ⅐h ؊1 ) for 6 h, and incorporation of label into whole blood GSH determined by GC͞MS selected ion monitoring. The fractional synthesis rate (mean ؎ SD; day -1 ) of whole blood GSH was 0.65 ؎ 0.13 for the adequate diet and 0.49 ؎ 0.13 for the SAA-free diet (P < 0.01). Whole blood GSH was 1,142 ؎ 243 and 1,216 ؎ 162 M for the adequate and SAA-free periods (P > 0.05), and the absolute rate of GSH synthesis was 747 ؎ 216 and 579 ؎ 135 mol⅐liter ؊1 ⅐day ؊1 , respectively (P < 0.05). Thus, a restricted dietary supply of SAA slows the rate of whole blood GSH synthesis and diminishes turnover, with maintenance of the GSH concentration in healthy subjects.
Estimates of substrate oxidation obtained from appearance of 13C or 14C from tracers in breath must be corrected for retention of labeled carbon in the body. We aimed to determine the effect of a defined experimental diet and metabolic status on recovery of infused Na [13C]bicarbonate in breath. Six healthy male subjects consumed an experimental diet for 7 days before receiving a continuous infusion of formula without tracer on day 8 and received either an intragastric (ig) or intravenous (iv) infusion of Na [13C]bicarbonate on day 9 or 11 during a 4-h postabsorptive (PA), 4-h continuously fed period. A trend toward increasing PA breath enrichment during the first 7 diet days approached statistical significance (P = 0.051), whereas breath enrichments measured 3 h postbreakfast were consistently higher than PA values throughout and did not change over the 7-day period. Breath enrichments during a 4-h continuous ig infusion of formula without tracer on day 8 rose 2.0 +/- 0.5 atom percent excess (APE).10(-3) above base line (P less than 0.001, ANOVA). In the tracer studies, breath enrichments were similar for the ig and iv routes of tracer infusion. For the ig infusion the fraction of infused Na [13C]bicarbonate recovered in breath as 13CO2 was 0.74 +/- 0.02 for the PA period and 0.79 +/- 0.02 for the fed period. For the iv infusion the fraction recovered was 0.70 +/- 0.04 for the PA period and 0.82 +/- 0.03 for the fed period. Fractional recoveries were not significantly different for ig and iv routes of administration but were different for PA and fed periods (P less than 0.0001, 2-way ANOVA). The fractional recoveries for the fed period obtained here were similar to the value 0.81 reported in a number of other studies. Recovery of tracer in breath increased linearly with O2 uptake and CO2 production, suggesting that factors affecting respiratory gas exchange may alter recovery. We conclude that the primary factor determining label recovery is the immediate and recent nutritional status of the host.
Inflammation and apoptosis develop in skeletal muscle after major trauma, including burn injury, and play a pivotal role in insulin resistance and muscle wasting. We and others have shown that inducible nitric oxide synthase (iNOS), a major mediator of inflammation, plays an important role in stress (e.g., burn)-induced insulin resistance. However, it remains to be determined how iNOS induces insulin resistance. Moreover, the interrelation between inflammatory response and apoptosis is poorly understood, although they often develop simultaneously. Nuclear factor (NF)-κB and p53 are key regulators of inflammation and apoptosis, respectively. Sirt1 inhibits p65 NF-κB and p53 by deacetylating these transcription factors. Recently, we have shown that iNOS induces S-nitrosylation of Sirt1, which inactivates Sirt1 and thereby increases acetylation and activity of p65 NF-κB and p53 in various cell types, including skeletal muscle cells. Here, we show that iNOS enhances burn-induced inflammatory response and apoptotic change in mouse skeletal muscle along with S-nitrosylation of Sirt1. Burn injury induced robust expression of iNOS in skeletal muscle and gene disruption of iNOS significantly inhibited burn-induced increases in inflammatory gene expression and apoptotic change. In parallel, burn increased Sirt1 S-nitrosylation and acetylation and DNA-binding capacity of p65 NF-κB and p53, all of which were reversed or ameliorated by iNOS deficiency. These results indicate that iNOS functions not only as a downstream effector but also as an upstream enhancer of burn-induced inflammatory response, at least in part, by Sirt1 S-nitrosylation-dependent activation (acetylation) of p65 NF-κB. Our data suggest that Sirt1 S-nitrosylation may play a role in iNOS-mediated enhanced inflammatory response and apoptotic change, which, in turn, contribute to muscle wasting and supposedly to insulin resistance after burn injury.
Despite significant advances in burn resuscitation and wound care over the past 30 years, morbidity and mortality from thermal injury remain high. Limited donor skin in severely burned patients hinders effective wound excision and closure, leading to infectious complications and prolonged hospitalizations. Even with large-volume fluid resuscitation, the systemic inflammatory response syndrome compromises end-organ perfusion in burn patients, with resultant multiorgan failure. Stem cells, which enhance wound healing and counteract systemic inflammation, now offer potential therapies for these challenges. Through a review of the literature, this article seeks to illustrate applications of stem cell therapy to burn care and to highlight promising areas of research.
In healthy adult men adapted to a diet/exercise regimen for 6 days, the effects of small, frequent meals supplying daily protein intakes of 1 ( n = 8) or 2.5 g ⋅ kg−1 ⋅ day−1( n = 6) on leucine oxidation, urea production, and whole body protein synthesis (PS) and degradation (PD) have been compared with the use of a 24-h continuous intravenous [13C]leucine and [15N,15N]urea infusion protocol. Two 90-min periods of exercise (∼50% maximal O2 consumption) were included during the fasting and the fed periods of the 24-h day. Subjects were determined to be at approximate energy, nitrogen, and leucine balances on both diets. Increased protein intake raised the urea production rate; the absolute rate of urea hydrolysis was the same on both diets. When the first-pass splanchnic uptake of leucine was taken to be 25% of intake, PS was stimulated by feeding (after an overnight fast) at both protein intake levels ( P < 0.05 and P < 0.01), whereas PD declined significantly ( P < 0.01) at both protein levels. Protein gain at a high protein intake appears to be the result of both a stimulation of PS and a marked decline in PD, whereas at a less generous intake, the gain appears to be a result of a fall in PD with a less evident change in PS. Exercise moderately decreased PS during and/or immediately after exercise at each protein level, and there was a postexercise-induced increase ( P < 0.01) in PD, which was more dramatic when feeding was at the higher protein intake level.
The role of the splanchnic region (Sp) in whole body leucine metabolism was assessed in six chronically catheterized fasting mongrel dogs and in eight dogs during constant enteral feeding of a complete amino acid solution (0.24 g.kg-1.h-1). We used primed continuous intravenous infusions of L-[1-13C,15N]leucine and L-[1-14C]leucine and measurements of arteriovenous isotope and leucine balance across the gut, liver, and Sp. In the fasted condition, 3.5% of arterial leucine supply was oxidized in the Sp, accounting for 13% of total body leucine oxidation, with 10% by liver. With amino acid feeding 1) leucine carbon and nitrogen fluxes and oxidation were increased (P less than 0.01) at the whole body level; 2) the percent of whole body leucine oxidation occurring in the Sp and liver increased (P less than 0.01) to 41 and 27%, respectively; 3) fractional metabolic utilization of leucine delivered to the Sp was reduced (P less than 0.01) from 47 to 35%; 4) the deamination rate of leucine in the gut was increased (P less than 0.05), along with an increased reamination rate of alpha-ketoisocaproic acid in the Sp (P less than 0.05). These findings reveal that the Sp accounts for a small fraction of whole body leucine oxidation during the fasting condition, but it plays a quantitatively important role in total body leucine oxidation during amino acid feeding; the gut and liver play cooperative roles in controlling leucine supply to peripheral tissues.
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