Effect of intermittent glutamine supplementation on skeletal muscle is not long-lasting in very old rats.

BACKGROUND AND OBJECTIVE: Muscle is the major site for glutamine synthesis via glutamine synthetase (GS). This enzyme is increased 1.5-2 fold in 25-27-mo rats and may be a consequence of aging-induced stress. This stimulation is similar to the induction observed following a catabolic state such as glucocorticoid treatment (6 to 24 months). Although oral glutamine supply regulates the plasma glutamine level, nothing is known if this supplementation is interrupted before the experiment. DESIGN: Adult (8-mo) and very old (27-mo) female rats were exposed to intermittent glutamine supplementation for 50 % of their age lifetime. Treated rats received glutamine added to their drinking water and control rats water alone but the effect of glutamine supplementation was only studied 15 days after the last supplementation. RESULTS: Glutamine pretreatment discontinued 15 days before the experiment increased plasma glutamine to ~ 0.6 mM, a normal value in very old rats. However, it failed to decrease the up-regulated GS activity in skeletal muscle from very old rats. CONCLUSION: Our results suggest that long-term treatment with glutamine started before advanced age but discontinued 15 days before rat sacrifice is effective in increasing plasma glutamine to recover basal adult value and in maintaining plasma glutamine in very old rats, but has no long-lasting effect on the GS activity of skeletal muscle with advanced age.

The role of adrenal hormones in the response of glutamine synthetase to fasting in adult and old rats.

BACKGROUND & AIMS: During fasting, skeletal muscle exports increased amounts of glutamine (Gln) while increasing the production of this amino acid by glutamine synthetase (GS) in order to maintain the intramuscular Gln pool. Glucocorticoid hormones are believed to be the principal mediators of GS induction during stress conditions. The aim of this study was to evaluate (1) the effect of fasting on GS activity and expression in skeletal muscle during aging and consequently, (2) the role of glucocorticoids in fasting-induced GS activity. METHODS: Male Wistar rats (6-, 22-month old) were fasted for 5 days and both the activity and expression of GS were measured in tibialis anterior muscle. To better demonstrate the role of glucocorticoids in the response of GS to fasting, we suppressed their action by RU38486 administration (a potent glucocorticoid antagonist) and their production by adrenalectomy in fed and fasted rats. RESULTS: An increase in fasting-induced GS activity was observed in skeletal muscles from both adult and aged rats. Adrenalectomy, but surprisingly not RU38486, suppressed the fasting-induced increase in GS activity and expression. CONCLUSION: The data clearly show that the GS responsiveness to fasting was not modified by aging in skeletal muscle.

Effects of glutamine deprivation on protein synthesis in a model of human enterocytes in culture.

To assess the effect of glutamine availability on rates of protein synthesis in human enterocytes, Caco-2 cells were grown until differentiation and then submitted to glutamine deprivation produced by exposure to glutamine-free medium or methionine sulfoximine [L-S-[3-amino-3-carboxypropyl]-S-methylsulfoximine (MSO)], a glutamine synthetase inhibitor. Cells were then incubated with (2)H(3)-labeled leucine with or without glutamine, and the fractional synthesis rate (FSR) of total cell protein was determined from (2)H(3)-labeled enrichments in protein-bound and intracellular free leucine measured by gas chromatography-mass spectrometry. Both protein FSR (28 +/- 1.5%/day) and intracellular glutamine concentration (6.1 +/- 0.6 micromol/g protein) remained unaltered when cells were grown in glutamine-free medium. In contrast, MSO treatment resulted in a dramatic reduction in protein synthesis (4.6 +/- 0.6 vs. 20.2 +/- 0.8%/day, P < 0.01). Supplementation with 0.5-2 mM glutamine for 4 h after MSO incubation, but not with glycine nor glutamate, restored protein FSR to control values (24 +/- 1%/day). These results demonstrate that in Caco-2 cells, 1) de novo glutamine synthesis is highly active, since it can maintain intracellular glutamine pool during glutamine deprivation, 2) inhibition of glutamine synthesis is associated with reduced protein synthesis, and 3) when glutamine synthesis is depressed, exogenous glutamine restores normal intestinal FSR. Due to the limitations intrinsic to the use of a cell line as an experimental model, the physiological relevance of these findings for the human intestine in vivo remains to be determined.

Lack of effect of ageing on acetate oxidation in rat skeletal muscle during starvation: a (13)C NMR study.

Acetate oxidation was examined by (13)C nuclear magnetic resonance in skeletal muscle from adult and old rats. Rats fasted for 5 days were perfused with [2-(13)C]acetate over 2 h, and muscle extracts were analyzed for [(13)C]glutamate isotopomers. This study shows that approximately 80 % of acetyl-coenzyme A entering the tricarboxylic cycle was derived from substrate infusion in both adult and old rats, and that the flux through anaplerotic pathways was approximately 21 % of the flux through citrate synthase. These data demonstrate that skeletal muscle from adult and old rats oxidizes the same proportion of exogenous acetate.

pH is regulated differently by glucose in skeletal muscle from fed and starved rats: a study using 31P-NMR spectroscopy.

The aim of this study was to determine whether exogenous glucose metabolism influences the pH in superfused EDL muscle from growing rats fed or starved for 48 h (body weight 55 and 45 g, respectively). Energy state and intracellular pH of muscle were repeatedly monitored by 31P-nuclear magnetic resonance spectroscopy (31P-NMRS); glycogen and other energy metabolites were assayed enzymatically in muscle extracts at the end of the experiment. In EDL muscles from starved rats superfused with glucose for 4 h, intracellular pH was elevated (7-7.3), lactate concentration low, glycogen repletion very intense and citrate synthase activity high. We conclude that glucose was routed mainly toward both oxidative phosphorylation and glycogen synthesis in EDL muscles after food deprivation of rats. In contrast, the major pathway in muscles from fed rats may be glycolysis because the glycogen pool remained constant throughout the experiment. The additional and minor pH component (in the range of 6.5 to 6.8) seen in muscles from fed rats, even in the presence of exogenous glucose, might be due to impaired glucose utilization because this component appears also in muscles from starved rats superfused without glucose or with a nonmetabolizable analog of glucose. Consequently, direct pH measurement by 31P-NMR may be considered to be a precise criterion for evaluation of differences in metabolic potentialities of muscle studied ex vivo in relation to the nutritional state of rats.

Contribution of proteolysis and de novo synthesis to alanine production in diabetic rat skeletal muscle: a 15N/1H nuclear magnetic resonance study.

To assess the role of leucine as a precursor of alanine alpha-amino nitrogen in skeletal muscle during diabetes, extensor digitorum longus muscles from control (n = 7 experiments) and streptozotocin-diabetic rats (n = 8 experiments) were isolated and superfused with [15N]leucine (3 mmol/l) in the presence of glucose (10 mmol/l) for 2 h. Muscle perchloric acid extraction was performed at the end of superfusion in order to quantify newly synthesized alanine by 15N/1H nuclear magnetic resonance. Release of [15N]alanine in the superfusion medium was also measured. The pool of newly synthesized [15N]alanine was significantly increased (approximately 40%) in extensor digitorum longus muscles from streptozotocin-diabetic rats. Whereas a significant enhancement of total alanine release from muscle was induced by diabetes (20%), only a slight increase in [15N]alanine release was detectable under our experimental conditions. Consequently, we conclude that streptozotocin-diabetes in growing rats induces in skeletal muscle: 1) an increase in nitrogen exchange between leucine and alanine leading to newly synthesized [15N]alanine; and 2) an increase of total alanine release from muscle originating from both proteolysis and de novo synthesis.

Measurement of glutamine synthetase activity in rat muscle by a colorimetric assay.

Glutamine synthetase catalyses the formation of L-Gln from L-Glu and NH4+. This enzyme also exerts a glutamyl-transferase activity that produces gamma-glutamyl-hydroxamate from Gln and hydroxylamine. This gamma-glutamyl-transfer reaction can be used to determine glutamine synthetase activity by colorimetric assay. This method has never been applied to rat muscle. The aim of this work was to study and optimize the glutamine synthetase assay conditions in rat muscle. Enzyme activity was linear with time of incubation (30 min at 37 degrees C) and linear with enzyme concentration in the incubation medium. The method was specific. In addition, this assay correlated well with a radiometric assay (y = 0.76x + 340, where x and y are the glutamine synthetase activities measured by radiometry and colorimetry respectively; r = 0.94; P = 0.05). Finally, no glutamine synthetase activity was found in muscles of rats treated with methionine sulfoximine, an inhibitor of glutamine synthetase, and activity dramatically rose in muscles from rats treated with dexamethasone, an activator of glutamine synthetase (in extensor digitorum longus: 2717 +/- 54 nmol/min/g protein in dexamethasone-treated rats versus 1228 +/- 114 nmol/min/g protein in control rats, P < 0.0001). In conclusion, the method presented here is accurate and reliable for measurement of glutamine synthetase activity in muscles.

Glutamine synthetase induction by glucocorticoids is preserved in skeletal muscle of aged rats.

Glutamine synthetase (GS) is a glucocorticoid-inducible enzyme that has a key role for glutamine synthesis in muscle. We hypothesized that the glucocorticoid induction of GS could be altered in aged rats, because alterations in the responsiveness of some genes to glucocorticoids were reported in aging. We compared the glucocorticoid-induced GS in fast-twitch and slow-twitch skeletal muscles (tibialis anterior and soleus, respectively) and heart from adult (age 6-8 mo) and aged (age 22 mo) female rats. All animals received dexamethasone (Dex) in their drinking water (0.77 +/- 0.10 and 0.80 +/- 0.08 mg/day per adult and aged rat, respectively) for 5 days. Dex caused an increase in both GS activity and GS mRNA in fast-twitch and slow-twitch skeletal muscles from adult and aged rats. In contrast, Dex increased GS activity in heart of adult rats, without any concomitant change in GS mRNA levels. Furthermore, Dex did not affect GS activity in aged heart. Thus the responsiveness of GS to an excess of glucocorticoids is preserved in skeletal muscle but not in heart from aged animals.

Coordinate activation of lysosomal, Ca 2+-activated and ATP-ubiquitin-dependent proteinases in the unweighted rat soleus muscle.

Nine days of hindlimb suspension resulted in atrophy (55%) and loss of protein (53%) in rat soleus muscle due to a marked elevation in protein breakdown (66%, P < 0.005). To define which proteolytic system(s) contributed to this increase, soleus muscles from unweighted rats were incubated in the presence of proteolytic inhibitors. An increase in lysosomal and Ca 2+-activated proteolysis (254%, P < 0.05) occurred in the atrophying incubated muscles. In agreement with the measurements in vitro, cathepsin B, cathepsins B + L and m-calpain enzyme activities increased by 111%, 92% and 180% (P < 0.005) respectively in the atrophying muscles. Enhanced mRNA levels for these proteinases (P < 0.05 to P < 0.001) paralleled the increased enzyme activities, suggesting a transcriptional regulation of these enzymes. However, the lysosomal and Ca 2+-dependent proteolytic pathways accounted for a minor part of total proteolysis in both control (9%) and unweighted rats (18%). Furthermore the inhibition of these pathways failed to suppress increased protein breakdown in unweighted muscle. Thus a non-lysosomal Ca 2+-independent proteolytic process essentially accounted for the increased proteolysis and subsequent muscle wasting. Increased mRNA levels for ubiquitin, the 14 kDa ubiquitin-conjugating enzyme E2 (involved in the ubiquitylation of protein substrates) and the C2 and C9 subunits of the 20 S proteasome (i.e. the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates) were observed in the atrophying muscles (P < 0.02 to P < 0.001). Analysis of C9 mRNA in polyribosomes showed equal distribution into both translationally active and inactive mRNA pools, in either unweighted or control rats. These results suggest that increased ATP-ubiquitin-dependent proteolysis is most probably responsible for muscle wasting in the unweighted soleus muscle.

Use of superfused rat skeletal muscle for metabolic studies: assessment of pH by 31P n.m.r.

We developed a muscle superfusion system suitable for metabolic studies of small isolated rat muscle ex vivo in real time and in a non-destructive manner by n.m.r. spectroscopy. In order to determine biochemical stability of superfused extensor digitorum longus (EDL) muscle (from fasted 45 and 100 g rats), the energy state and the pH of muscle were continuously monitored by 31P n.m.r. spectroscopy. ATP and phosphocreatine remained stable during 2 h whatever the muscle size (20 or 45 mg). Neither metabolite was a sensitive probe of possible metabolic compartmentation within muscle under our experimental conditions. By contrast, the chemical shift of Pi by its sensitivity to pH was a discriminant factor in the assessment of muscle stability. Indeed, heterogeneity of pH was observed only in the 45 mg EDL muscle resulting from a core region with loss of glycogen. Together, these observations suggest deviations of energy metabolism to supply ATP. Consequently, pH may be considered as a new real-time criterion for monitoring a metabolic heterogeneity due to changes in energy metabolism of muscle preparations ex vivo.

Heteronuclear 15N/1H spin echo NMR: an in vitro quantitative analysis of leucine transamination in rat skeletal muscle.

Detection of the 15N nucleus in studies of the metabolism of branched-chain amino acids was carried out by recording the 1H nuclear magnetic resonance (NMR) spectrum through the effect of the 15N-1H coupling. The Selective Excitation Unit performed a 90 degrees selective proton pulse to overcome the strong water signal and baseline distorsion. In order to obtain quantitative measurement, the leucine beta protons and the valine (internal reference) beta protons coupled to 15N nucleus were simultaneously detected. This NMR method was tested on muscle homogenate incubated with [15N] leucine (approximately 3 mumoles/g). The supernatant was directly observed by NMR. The sensitivity of this indirect method was found to be far higher than direct observation of the 15N signals by 15N NMR.