Synergistic effects of caloric restriction with maintained protein intake on skeletal muscle performance in 21-month-old rats: a mitochondria-mediated pathway.

Caloric restriction (CR) delays the onset of age-related mitochondrial abnormalities but does not prevent the decline in ATP production needed to sustain muscle protein fractional synthesis rate (FSR) and contractile activity. We hypothesized that improving mitochondrial activity and FSR using a CR diet with maintained protein intakes could enhance myofibrillar protein FSR and consequently improve muscle strength in aging rats. Wistar rats (21 months old) were fed either an ad libitum (AL), 40% protein-energy restricted (PER) or 40% AL-isonitrogenous energy restricted (ER) diet for 5 months. ATP production, electron transport chain activity, reactive oxygen species (ROS) generation, protein carbonyl content and FSR were determined in both tibialis anterior (TA) and soleus muscle mitochondria. Myosin and actin FSR and grip force were also investigated. The ER diet led to improved mitochondrial activity and ATP production in the TA and soleus muscles in comparison with PER. Furthermore, mitochondrial FSR in the TA was enhanced under the ER diet but diminished under the PER. Mitochondrial protein carbonyl content was decreased by both the ER and PER diets. The ER diet was able to improve myosin and actin FSR and grip force. Therefore, the synergistic effects of CR with maintained protein intake may help to limit the progression of sarcopenia by optimizing the turnover rates and functions of major proteins in skeletal muscle.

Estimating body composition in children with Duchenne muscular dystrophy: comparison of bioelectrical impedance analysis and skinfold-thickness measurement.

BACKGROUND: Duchenne muscular dystrophy (DMD) is often associated with obesity, which worsens the handicap early in the course of the disease. Nutritional assessment, however, can be difficult and often misleading in DMD. OBJECTIVE: Two methods of estimating body composition in DMD, skinfold-thickness (ST) measurement and bioelectrical impedance analysis (BIA), were compared with a reference method, labeled water dilution (WD). DESIGN: Body composition was estimated by using ST measurements and BIA (50 kHz, 800 mAmp), as well as the WD method (1 mL H2(18)O/kg) in 11 DMD patients with a mean (+/-SD) age of 10.0 +/- 2.5 y. RESULTS: When compared with the WD method, ST measurement significantly (P < 0.01) overestimated fat-free mass (FFM) (mean +/- SD ST: 24.5 +/- 5.9 kg; mean +/- SD WD: 18.2 +/- 2.5 kg), which led to an underestimation of the percentage of fat mass (%FM) (ST: 23.3 +/- 10.4%; WD: 40.1 +/- 17.1%; P < 0.05). In contrast, estimates obtained with BIA (FFM: 21.5 +/- 4.5 kg; %FM: 31.3 +/- 13.9%) did not differ from those obtained with WD. The difference from the reference method was less for BIA (mean: 3.3 kg; 95% CI: 0.8, 4.9 kg) than for ST (6.3 kg; 2.2, 8.6 kg). WD and BIA defined 73% and 55%, respectively, of the children as obese (%FM associated with body mass index cutoffs for obesity), whereas ST measurements defined 9% as obese (P < 0.01). CONCLUSIONS: Body-composition estimates by BIA are closer to those by WD than are those by ST measurement. Early detection of fat accumulation and longitudinal monitoring of nutritional care are 2 relevant applications of BIA to prevent obesity and hence lessen the burden of DMD.

Impaired anabolic response of muscle protein synthesis is associated with S6K1 dysregulation in elderly humans.

Age-related loss of muscle protein may involve a decreased response to anabolic factors of muscle protein synthesis through dysregulation of translation factors. To verify this hypothesis, we simultaneously investigated muscle protein synthesis and expression of some factors implicated in insulin signal transduction during hyperinsulinemia and hyperaminoacidemia in 6 young (25+/-1 year; mean+/-sem) and 8 elderly subjects (72+/-2 year). Incorporation of L-[1-13C] leucine in muscle proteins (fractional synthesis rate, FSR) was measured in vastus lateralis, before and during a euglycemic hyperinsulinemic hyperaminoacidemic clamp, together with Western blot analysis of protein kinase B (PKB), mTOR, 4E-BP1, and S6K1 phosphorylation. In basal state, muscle protein FSR was reduced in elderly in comparison with young subjects (0.061+/-0.004% per hour) vs 0.082+/-0.010% per hour, elderly vs. young, P<0.05). During clamp, muscle protein FSR was stimulated in young (0.119+/-0.006% per hour; P<0.05), but this response was significantly lower in elderly subjects (0.084+/-0.005% per hour, P<0.05 vs young subjects). Phosphorylation of PKB, mTOR, and 4E-BP1 were similarly increased by insulin and amino acid in both groups, except for S6K1 phosphorylation, which was not stimulated in elderly subjects. In conclusion, 1) response of muscle protein synthesis to insulin and amino acid is impaired in elderly humans; 2) a defect in S6K1 pathway activation may be responsible for this alteration. This modification is a mechanistic basis of sarcopenia development during aging.

Insulin regulates protein synthesis rate in leukocytes from young and elderly healthy humans.

Immune response is dependent on synthesis and secretion of various proteins. Hence, in vivo determination of protein synthesis rate in leukocytes may reflect the functional activity of these cells under conditions of immune deficiency such as aging. Because (i) insulin stimulates protein turnover in various tissues and (ii) elderly people often suffer from a decrease in insulin sensitivity, we sought to determine the effect of physiological hyperinsulinemia (0.7mU/kg fat free mass/min) on protein synthesis rate in peripheral blood mononuclear cells (PBMCs) and polymorphonuclear neutrophils (PMNs) from 8 adult (24+/-6yr) and 9 elderly (69+/-4yr) healthy subjects under strict euglycemia (0.9g/l). The body mass index was significantly higher in elderly volunteers compared to the younger ones (P<0.05) whereas insulin sensitivity was lower as shown by either the M value (P<0.05) or the glucose disposal rate (GDR)/insulinemia ratio (P<0.05). The density of the insulin receptor at PBMCs and PMNs surfaces was not altered by either age or insulin infusion. In comparison with the baseline values, insulin highly stimulated the fractional synthesis rate (FSR) of mixed PBMCs (4.12+/-0.56%/d vs. 6.94+/-0.70%/d, P<0.05) and PMNs (1.05+/-0.28%/d vs. 2.44+/-0.41%/d, P<0.05) proteins. No age effect was observed either during the baseline (PBMCs: 4.31+/-0.30%/d, PMNs: 1.55+/-0.28%/d) period or after insulin infusion (PBMCs: 6.86+/-0.69%/d, PMNs: 2.76+/-0.47%/d). We conclude that insulin is able to stimulate protein synthesis in leukocyte populations independently of the age of the subjects.

Whole body protein breakdown is less inhibited by insulin, but still responsive to amino acid, in nondiabetic elderly subjects.

Responses of whole body glucose disposal (GDR) and protein breakdown (PB) to physiological insulin levels are altered in nondiabetic elderly subjects. Amino acids enhance inhibition of PB by insulin in young subjects. We hypothesized that addition of amino acid to insulin may improve the defect in PB regulation by insulin in elderly people. Therefore, we investigated the effect of hyperinsulinemia combined to either euaminoacidemia (EuAA) or hyperaminoacidemia (HyperAA) on GDR and PB, using isotopic dilution of D-[6,6-2H2]glucose and L-[1-13C]leucine, in young (mean +/- SEM, 24.4 +/- 0.8 yr) and elderly (70.2 +/- 0.7 yr) subjects. GDR was lower in elderly than in young subjects in all situations (P < 0.05). Despite a greater inhibition with HyperAA, PB was less inhibited in elderly than in young subjects during both clamps (ratio between change over basal PB and change over basal insulinemia, -0.014 +/- 0.002 vs. -0.024 +/- 0.003 in EuAA and -0.022 +/- 0.002 vs. -0.036 +/- 0.003 micromol/ml.microU/kg fat-free mass.min in HyperAA; elderly vs. young, P < 0.05). In conclusion, in nondiabetic elderly subjects, PB is less inhibited by insulin with either basal or high amino acid concentrations. Addition of amino acid potentiates insulin-induced suppression of PB in both groups to the same extent, suggesting a specific dysregulation of PB by insulin with age.

Human albumin synthesis is increased by an ultra-endurance trial.

PURPOSE: The purpose of this study was to determine whether an ultra-endurance event is a strong stimulus to increase albumin synthesis involved in the process of intravascular albumin mass increase associated with transient hypervolemia. METHODS: The albumin synthetic rate was measured in six young men, 3 d before (C) and on the 1st (R1) and 8th (R8) days of the recovery from an ultra-endurance trial (5 h daily for 4 d). Albumin fractional (FSR) and absolute (ASR) synthetic rate were determined using a primed-constant infusion of [1(-13) C] leucine. Plasma volume (PV) using Evans Blue dye dilution and total body water (TBW) using bioelectrical impedance analysis were measured on C, R1, and R8. RESULTS: On R1 as compared with C: 1). PV (+23.3 +/- 3.2%; P

Effect of high altitude on protein metabolism in Bolivian children.

In Bolivia, malnutrition in children is a major health problem that may be caused by inadequate protein, energy, and micronutrient intake; exposure to bacterial and parasitic infections; and life in a multistress environment (high altitude, cold, cosmic radiation, low ambient humidity). However, no data on protein absorption and utilization at high altitude were available. Therefore, we evaluated the effect of altitude on protein metabolism in Bolivian children. We measured protein utilization using leucine labeled with a stable isotope ((13)C) in two groups of healthy prepubertal children matched for age. Group 1 (n = 10) was examined at high altitude (HA) in La Paz (3600 m), and group 2 (n = 10) at low altitude (LA) in Santa Cruz (420 m). The nutritional status did not differ between groups but, as was to be expected, the HA group had higher hemoglobin concentration than the LA group. The children consumed casein that was intrinsically labeled with L-(1-(13)C) leucine and expired (13)CO(2) was analyzed. Samples of expired air were measured by isotope ratio mass spectrometer in Clermont-Ferrand. It was found that cumulative leucine oxidation ((13)CO(2)) at 300 min after ingestion was 19.7 +/- 4.9% at HA and 25.2 +/- 3.2% at LA. These results showed that protein absorption and/or utilization is significantly affected by altitude.

Effect of equilibrated hydration changes on total body water estimates by bioelectrical impedance analysis.

The present study was performed to determine how equilibrated fluctuations in hydration affected the validity of bioelectrical impedance analysis (BIA) for body composition assessment. Total body water (TBW) expansion was induced by a 4 d endurance trial and the subsequent water loss was obtained over the recovery period. Twelve healthy men exercised on a cycle and treadmill alternately for 5 h/d over 4 d at moderate intensity. TBW, fat mass (FM) and fat-free mass (FFM) were assessed 3 d before the trial (control), and on the first and eighth day of recovery (R1 and R8 respectively). TBW was evaluated by (2)H dilution (TBW2H) as a reference method and by BIA (TBWBIA) at 100 kHz at the same time. TBW2H increased significantly between the control day and R1 by 1.87 (sd 1.11) litres (P=0.005) and TBWBIA by 1.38 (sd 1.56) litres (P=0.009). Both values returned to the control level on R8. For each period, TBW2H and TBWBIA did not differ significantly and were correlated (r(2) 0.85, P=0.0004 for the control day; r(2) 0.63, P=0.03 for R1; r(2) 0.75, P=0.02 for R8). Plasma Na concentration and osmolality did not differ between the control day, R1 and R8. FFM gain (1208 (sd 1983) g) and FM loss (-1168 (sd 906) g) between the control day and R1 were followed by a FFM decrease (-624 (sd 1281) g) and a FM increase (860 (sd 1212) g) between R1 and R8. As expected, these FFM and FM changes were significantly correlated with TBW variations. The present results provide evidence that BIA may be a useful method for estimating TBW when fluid shifts are equilibrated and electrolyte concentrations are unchanged. However, it is not a valid technique for assessing FM and FFM under these conditions.

Protein synthesis rates of human PBMC and PMN can be determined simultaneously in vivo by using small blood samples.

Immune cell functions can be evaluated in vivo by measuring their specific protein fractional synthesis rates (FSR). Using stable isotope dilution techniques, we describe a new method allowing simultaneous in vivo assessment of FSR in two leukocyte populations in healthy human subjects, using small blood samples. Peripheral blood mononuclear cell (PBMC) and polymorphonuclear neutrophil (PMN) FSR were measured during primed continuous intravenous infusion of l-[1-(13)C]leucine. Immune cells from 6 ml of whole blood were isolated by density gradient centrifugation. In a first study, we calculated the FSR using plasma [(13)C]leucine or alpha-[(13)C]ketoisocaproate (KIC) enrichments as precursor pools. In a second study, we compared protein FSR in leukocytes, using enrichments of either intracellular or plasma free [(13)C]leucine as immediate precursor pools. The present approach showed a steady-state enrichment of plasma and circulating immune cell free [(13)C]leucine precursor pools. The linearity of labeled amino acid incorporation rate within mixed PBMC and PMN proteins also was verified. Postabsorptive protein FSR was 4.09 +/- 0.39%/day in PBMC and 1.44 +/- 0.08%/day in PMN when plasma [(13)C]KIC was the precursor pool. The difference between PBMC and PMN FSR was statistically significant, whatever the precursor pool used, suggesting large differences in their synthetic activities and functions. Use of the plasma [(13)C]KIC pool led to an underestimation of leukocyte FSR compared with the intracellular pool (PBMC: 6.04 +/- 0.94%/day; PMN: 2.98 +/- 0.30%/day). Hence, the intracellular free amino acid pool must be used as precursor to obtain reliable results. In conclusion, it is possible to assess immune cell metabolism in vivo in humans by using small blood samples to directly indicate their metabolic activity in various clinical situations and in response to regulating factors.

The rate of protein digestion affects protein gain differently during aging in humans.

In young men ingesting protein meals, slowly digested proteins (caseins: CAS) induce a higher protein gain than those that are rapidly digested (whey proteins: WP). Our aim was to assess whether or not this is true in elderly men receiving mixed meals. The effects of meals containing either CAS or two different amounts of WP (WP-iN: isonitrogenous with CAS, or WP-iL: providing the same amount of leucine as CAS) on protein metabolism (assessed by combining oral and intravenous leucine tracers) were compared in nine healthy, elderly (mean +/- S.E.M. age 72 +/- 1 years) and six young men (24 +/- 1 years). In both age groups, WP-iL and WP-iN were digested faster than CAS (P < 0.001, ANOVA). Proteolysis was inhibited similarly whatever the meal and age groups (P = NS). Protein synthesis was higher with WP-iN than with CAS or WP-iL (P < 0.01), irrespective of age (P = NS). An age-related effect (P < 0.05) was found with postprandial leucine balance. Leucine balance was higher with CAS than with WP-iL (P < 0.01) in young men, but not in elderly subjects (P = NS). In isonitrogenous conditions, leucine balance was higher with WP-iN than with CAS (P < 0.001) in both age groups, but the magnitude of the differences was higher in the elderly men (P = 0.05). In conclusion, during aging, protein gain was greater with WP (rapidly digested protein), and lower with CAS (slowly digested protein). This suggests that a 'fast' protein might be more beneficial than a 'slow' one to limit protein losses during aging.