Effect of colon cancer and surgical resection on skeletal muscle mitochondrial enzyme activity in colon cancer patients: a pilot study.

BACKGROUND: Colon cancer (CC) patients commonly suffer declines in muscle mass and aerobic function. We hypothesised that CC would be associated with reduced muscle mass and mitochondrial enzyme activity and that curative resection would exacerbate these changes. METHODS: We followed age-matched healthy controls and CC patients without distant metastasis on radiological imaging before and 6 weeks after hemi-colectomy surgery. Body composition was analysed using dual energy X-ray absorptiometry. Mitochondrial enzyme activity and protein concentrations were analysed in vastus lateralis muscle biopsies. RESULTS: In pre-surgery, there were no differences in lean mass between CC patients and age-matched controls (46.1 + 32.5 vs. 46.1 + 37.3 kg). Post-resection lean mass was reduced in CC patients (43.8 + 30.3 kg, P < 0.01). When comparing markers of mitochondrial function, the following were observed: pyruvate dehydrogenase (PDH) activity was lower in CC patients pre-surgery (P < 0.001) but normalized post-resection and cytochrome c oxidase and pyruvate dehydrogenase E2 subunit protein expression were lower in CC patients pre-surgery and not restored to control values post-resection (P < 0.001). Nuclear factor kappa-B, an inflammatory marker, was higher in CC patients pre-surgery compared to controls (P < 0.01), returning to control levels post-resection. CONCLUSION: Muscle mass was affected by surgery rather than cancer per se. PDH activity was however lower in cancer patients, suggesting that muscle mass and mitochondrial enzyme activity are not inextricably linked. This reduction in mitochondrial enzyme activity may well contribute to the significant risks of major surgery to which CC patients are exposed.

Effect of tumor burden and subsequent surgical resection on skeletal muscle mass and protein turnover in colorectal cancer patients.

BACKGROUND: Cachexia is a consequence of tumor burden caused by ill-defined catabolic alterations in muscle protein turnover. OBJECTIVE: We aimed to explore the effect of tumor burden and resection on muscle protein turnover in patients with nonmetastatic colorectal cancer (CRC), which is a surgically curable tumor that induces cachexia. DESIGN: We recruited the following 2 groups: patients with CRC [n = 13; mean ± SEM age: 66 ± 3 y; BMI (in kg/m(2)): 27.6 ± 1.1] and matched healthy controls (n = 8; age: 71 ± 2 y; BMI: 26.2 ± 1). Control subjects underwent a single study, whereas CRC patients were studied twice before and ~6 wk after surgical resection to assess muscle protein synthesis (MPS), muscle protein breakdown (MPB), and muscle mass by using dual-energy X-ray absorptiometry. RESULTS: Leg muscle mass was lower in CRC patients than in control subjects (6290 ± 456 compared with 7839 ± 617 g; P < 0.05) and had an additional decline after surgery (5840 ± 456 g; P < 0.001). Although postabsorptive MPS was unaffected, catabolic changes with tumor burden included the complete blunting of postprandial MPS (0.038 ± 0.004%/h in the CRC group compared with 0.065 ± 0.006%/h in the control group; P < 0.01) and a trend toward increased MPB under postabsorptive conditions (P = 0.09). Although surgical resection exacerbated muscle atrophy (-7.2%), catabolic changes in protein metabolism had normalized 6 wk after surgery. The recovery in postprandial MPS after surgery was inversely related to the degree of muscle atrophy (r = 0.65, P < 0.01). CONCLUSIONS: CRC patients display reduced postprandial MPS and a trend toward increased MPB, and tumor resection reverses these derangements. With no effective treatment of cancer cachexia, future therapies directed at preserving muscle mass should concentrate on alleviating proteolysis and enhancing anabolic responses to nutrition before surgery while augmenting muscle anabolism after resection.

Muscle protein synthetic responses to exercise: effects of age, volume, and intensity.

We explored the relationships between resistance exercise volume/intensity and muscle myofibrillar protein synthetic (MPS) responses in young and older men. In a crossover design, four groups of six young (24±6 years) and older (70±5 years) men performed two volumes of resistance exercise: either 40% one repetition maximum (1RM) (3 × 14, then 6 × 14 repetitions) or 75% 1RM (3 × 8, then 6 × 8 repetitions), such that at the same volume, work was identical between intensities. Muscle biopsies were taken 0, 1, 2, and 4hours after exercise to measure MPS via myofibrillar bound [1,2-(13)C(2)]leucine and indices of mammalian target of rapamycin signaling by immunoblotting. In younger men, doubling exercise volume produced limited added effects, whereas in older men, it resulted in greater MPS and p70S6 kinase (p70S6K(Thr389)) phosphorylation at both intensities, that is, MPS area under the curve: 75% (1× volume: 0.07±0.01 vs 2× volume: 0.14% ± 0.02% protein synthesized/4hours (p < .001). Doubling exercise volume is a valid strategy to maximize postexercise MPS in ageing.

Omega-3 polyunsaturated fatty acids augment the muscle protein anabolic response to hyperinsulinaemia-hyperaminoacidaemia in healthy young and middle-aged men and women.

Increased dietary LCn-3PUFA (long-chain n-3 polyunsaturated fatty acid) intake stimulates muscle protein anabolism in individuals who experience muscle loss due to aging or cancer cachexia. However, it is not known whether LCn-3PUFAs elicit similar anabolic effects in healthy individuals. To answer this question, we evaluated the effect of 8 weeks of LCn-3PUFA supplementation (4 g of Lovaza®/day) in nine 25-45-year-old healthy subjects on the rate of muscle protein synthesis (by using stable isotope-labelled tracer techniques) and the activation (phosphorylation) of elements of the mTOR (mammalian target of rapamycin)/p70S6K (p70 S6 kinase) signalling pathway during basal post-absorptive conditions and during a hyperinsulinaemic-hyperaminoacidaemic clamp. We also measured the concentrations of protein, RNA and DNA in muscle to obtain indices of the protein synthetic capacity, translational efficiency and cell size. Neither the basal muscle protein fractional synthesis rate nor basal signalling element phosphorylation changed in response to LCn-3PUFA supplementation, but the anabolic response to insulin and amino acid infusion was greater after LCn-3PUFA [i.e. the muscle protein fractional synthesis rate during insulin and amino acid infusion increased from 0.062±0.004 to 0.083±0.007%/h and the phospho-mTOR (Ser2448) and phospho-p70S6K (Thr389) levels increased by ∼50%; all P<0.05]. In addition, the muscle protein concentration and the protein/DNA ratio (i.e. muscle cell size) were both greater (P<0.05) after LCn-3PUFA supplementation. We conclude that LCn-3PUFAs have anabolic properties in healthy young and middle-aged adults.

Enhanced amino acid sensitivity of myofibrillar protein synthesis persists for up to 24 h after resistance exercise in young men.

We aimed to determine whether an exercise-mediated enhancement of muscle protein synthesis to feeding persisted 24 h after resistance exercise. We also determined the impact of different exercise intensities (90% or 30% maximal strength) or contraction volume (work-matched or to failure) on the response at 24 h of recovery. Fifteen men (21 ± 1 y, BMI = 24.1 ± 0.8 kg · m(-2)) received a primed, constant infusion of l-[ring-(13)C(6)]phenylalanine to measure muscle protein synthesis after protein feeding at rest (FED; 15 g whey protein) and 24 h after resistance exercise (EX-FED). Participants performed unilateral leg exercises: 1) 4 sets at 90% of maximal strength to failure (90FAIL); 2) 30% work-matched to 90FAIL (30WM); or 3) 30% to failure (30FAIL). Regardless of condition, rates of mixed muscle protein and sarcoplasmic protein synthesis were similarly stimulated at FED and EX-FED. In contrast, protein ingestion stimulated rates of myofibrillar protein synthesis above fasting rates by 0.016 ± 0.002%/h and the response was enhanced 24 h after resistance exercise, but only in the 90FAIL and 30FAIL conditions, by 0.038 ± 0.012 and 0.041 ± 0.010, respectively. Phosphorylation of protein kinase B on Ser473 was greater than FED at EX-FED only in 90FAIL, whereas phosphorylation of mammalian target of rapamycin on Ser2448 was significantly increased at EX-FED above FED only in the 30FAIL condition. Our results suggest that resistance exercise performed until failure confers a sensitizing effect on human skeletal muscle for at least 24 h that is specific to the myofibrillar protein fraction.

Carbohydrate does not augment exercise-induced protein accretion versus protein alone.

PURPOSE: We tested the thesis that CHO and protein coingestion would augment muscle protein synthesis (MPS) and inhibit muscle protein breakdown (MPB) at rest and after resistance exercise. METHODS: Nine men (age=23.0±1.9 yr, body mass index=24.2±2.1 kg·m) performed two unilateral knee extension trials (four sets×8-12 repetitions to failure) followed by consumption of 25 g of whey protein (PRO) or 25 g of whey protein plus 50 g of maltodextrin (PRO+CARB). Muscle biopsies and stable isotope methodology were used to measure MPS and MPB. RESULTS: The areas under the glucose and insulin curves were 17.5-fold (P<0.05) and 5-fold (P<0.05) greater, respectively, for PRO+CARB than for PRO. Exercise increased MPS and MPB (both P<0.05), but there were no differences between PRO and PRO+CARB in the rested or exercised legs. Phosphorylation of Akt was greater in the PRO+CARB than in the PRO trial (P<0.05); phosphorylations of Akt (P=0.05) and acetyl coA carboxylase-ß (P<0.05) were greater after exercise than at rest. The concurrent ingestion of 50 g of CHO with 25 g of protein did not stimulate mixed MPS or inhibit MPB more than 25 g of protein alone either at rest or after resistance exercise. CONCLUSIONS: Our data suggest that insulin is not additive or synergistic to rates of MPS or MPB when CHO is coingested with a dose of protein that maximally stimulates rates of MPS.

Muscle full effect after oral protein: time-dependent concordance and discordance between human muscle protein synthesis and mTORC1 signaling.

BACKGROUND: We previously showed that human muscle protein synthesis (MPS) increased during infusion of amino acids (AAs) and peaked at ≈120 min before returning to baseline rates, despite elevated plasma AA concentrations. OBJECTIVE: We tested whether a protein meal elicited a similar response and whether signaling responses that regulate messenger RNA translation matched MPS changes. DESIGN: Eight postabsorptive healthy men (≈21 y of age) were studied during 8.5 h of primed continuous infusion of [1,2-¹³C2]leucine with intermittent quadriceps biopsies for determination of MPS and anabolic signaling. After 2.5 h, subjects consumed 48 g whey protein. RESULTS: At 45-90 min after oral protein bolus, mean (± SEM) myofibrillar protein synthesis increased from 0.03 ± 0.003% to 0.10 ± 0.01%/h; thereafter, myofibrillar protein synthesis returned to baseline rates even though plasma essential AA (EAA) concentrations remained elevated (+130% at 120 min, +80% at 180 min). The activity of protein kinase B (PKB) and phosphorylation of eukaryotic initiation factor 4G preceded the rise of MPS and increases in phosphorylation of ribosomal protein kinase S6 (S6K1), and 4E-binding protein 1 (4EBP1) was superimposable with MPS responses until 90 min. However, although MPS decreased thereafter, all signals, with the exception of PKB activity (which mirrored insulin responses), remained elevated, which echoed the slowly declining plasma EAA profile. The phosphorylation of eukaryotic initiation factor 2α increased only at 180 min. Thus, discordance existed between MPS and the mammalian target of rapamycin complex 1 (mTORC1) and signaling (ie, S6K1 and 4EBP1 phosphorylation). CONCLUSIONS: We confirm our previous findings that MPS responses to AAs are transient, even with oral protein bolus. However, changes in MPS only reflect elevated mTORC1 signaling during the upswing in MPS.

Blunting of insulin inhibition of proteolysis in legs of older subjects may contribute to age-related sarcopenia.

BACKGROUND: Reduced postprandial muscle proteolysis is mainly due to increased insulin availability. Whether rates of proteolysis in response to low physiologic doses of insulin are affected by aging is unknown. OBJECTIVES: We tested the hypothesis that suppression of leg protein breakdown (LPB) by insulin is blunted in older subjects, together with blunted activation of Akt-protein kinase B (PKB). DESIGN: Groups of 8 young [mean (+/-SD) age: 24.5 +/- 1.8 y] and older (65.0 +/- 1.3 y) participants were studied during euglycemic (5 mmol/L), isoaminoacidemic (blood leucine approximately 120 micromol/L) clamp procedures at plasma insulin concentrations of approximately 5 and approximately 15 microIU/mL for 1.5 h. Leg amino acid balance, whole-leg protein turnover (as dilution of amino acid tracers), and muscle protein synthesis were measured with D(5)-phenylalanine and [1,2-(13)C(2)]leucine. The kinase activity of muscle Akt-PKB and the extent of phosphorylation of signaling proteins associated with the mTOR (mammalian target of rapamycin) pathway were measured before and after the clamp procedures. RESULTS: Basal LPB rates were not different between groups (66 +/- 11 compared with 51 +/- 10 nmol leucine x 100 mL leg(-1) x min(-1) and 30 +/- 5 compared with 24 +/- 4 nmol phenylalanine x 100 mL leg(-1) x min(-1) in young and older groups, respectively). However, although insulin at approximately 15 microIU/mL lowered LPB by 47% in the young subjects (P < 0.05) and abolished the negative leg amino acid balance, this caused only a 12% fall (P > 0.05) in the older group. Akt-PKB activity mirrored decreases in LPB. No differences were seen in muscle protein synthesis or associated anabolic signaling phosphoproteins. CONCLUSIONS: At moderate availability, the effect of insulin on LPB is diminished in older human beings, and this effect may be mediated through blunted Akt-PKB activation.

No major sex differences in muscle protein synthesis rates in the postabsorptive state and during hyperinsulinemia-hyperaminoacidemia in middle-aged adults.

Men have more muscle than women, but most studies evaluating sex differences in muscle protein metabolism have been unable to discern sexual dimorphism in basal muscle protein turnover rates in young and middle-aged adults. We hypothesized that the anabolic response to nutritional stimuli (i.e., amino acids and insulin) would be greater in young/middle-aged men than women. We therefore measured the rates of muscle protein synthesis (MPS) in 16 healthy individuals [8 men and 8 women, matched for age (mean +/- SE: 37.7 +/- 1.5 yr) and body mass index (25.2 +/- 0.7 kg/m2)] after an overnight fast (plasma insulin approximately 5 microU/ml and plasma phenylalanine approximately 60 microM) and during a hyperinsulinemic-hyperaminoacidemic-euglycemic clamp (plasma insulin approximately 28 microU/ml; plasma phenylalanine approximately 110 microM; plasma glucose approximately 5.4 mM). The rates of MPS were not different between men and women (ANOVA main effect for sex; P = 0.49). During the clamp, the rate of MPS increased by approximately 50% (P = 0.003) with no difference in the increases from basal values between men and women (+0.019 +/- 0.004 vs. +0.018 +/- 0.010%/h, respectively; P = 0.93). There were also no differences between men and women in the basal concentrations of muscle phosphorylated Akt(Ser473), Akt(Thr308), mTOR(Ser2448), and p70s6k(Thr389) or in the hyperinsulinemia-hyperaminoacidemia-induced increases in phosphorylation of those signaling elements (P > or = 0.25). We conclude that there are no major differences in the rate of MPS and its intracellular control during basal conditions and during hyperinsulinemia-hyperaminoacidema between young and middle-aged adult men and women.

Blunting of AICAR-induced human skeletal muscle glucose uptake in type 2 diabetes is dependent on age rather than diabetic status.

We demonstrated previously that, in healthy young men, 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR) stimulates human muscle 2-deoxyglucose (2DG) uptake without detectable activation of muscle AMP-activated protein kinase (AMPK) but with extracellular-regulated kinase 1/2 (ERK1/2) activation. We tested whether AICAR stimulates muscle 2DG uptake in healthy older patients with or without type 2 diabetes (T2D). Six healthy young subjects (23 +/- 3 yr, BMI 25 +/- 2 kg/m(-2); means +/- SE), eight older subjects (59 +/- 4 yr, BMI 28 +/- 2 kg/m(-2)), and eight subjects with T2D (62 +/- 4 yr, BMI 27 +/- 2 kg/m(-2)) received a 6-h 2DG infusion (prime 10 mg/kg, 6 mg.kg(-1).h(-1)) and AICAR (10 or 20 mg.kg(-1).h(-1)) from 3 to 6 h. Quadriceps biopsies were taken at 0, 3, and 6 h. We determined 1) 2DG uptake, 2) total AMPKalpha activity, AMPK, acetyl-CoA carboxylase (ACC), and AS160 phosphorylation, and 3) ERK1/2 phosphorylation. Ten milligrams per kilogram per hour AICAR increased 2DG uptake by 2.9 +/- 0.7-fold in young men (P < 0.001), 1.8 +/- 0.2-fold in older men (P < 0.01), and 1.6 +/- 0.1-fold in men with T2D; 20 mg.kg(-1).h(-1) AICAR increases were 2.5 +/- 0.1-fold (older men, P < 0.001) and 2.2 +/- 0.2-fold (men with T2D, P < 0.001). At 3-h AMPK activity and AMPK, ACC and AS160 phosphorylation were unchanged, but ERK1/2 phosphorylation increased at both AICAR doses. The fold changes of ERK1/2 phosphorylation and 2DG uptake closely correlated (R(2) = 0.55, P = 0.003). AICAR stimulates muscle 2DG uptake in T2D to the same extent as in healthy age-matched controls, but there is an age-related reduction.

Immobilization induces anabolic resistance in human myofibrillar protein synthesis with low and high dose amino acid infusion.

We tested the hypothesis that increasing blood amino acid (AA) availability would counter the physical inactivity-induced reduction in muscle protein synthesis. We determined how 14 days of unilateral knee immobilization affected quadriceps myofibrillar protein synthesis (MPS) in young healthy subjects (10 men, 2 women, 21 +/- 1 years; 80.2 +/- 4.0 kg, mean +/- S.E.M.) in the post-absorptive state and after infusing AA (10% Primene) at low or high doses (43 and 261 mg kg(-1) h(-1)). Muscle cross-sectional area (MRI) and peak isometric torque declined in the immobilized leg (-5.0 +/- 1.2% and -25 +/- 3%, respectively, both P < 0.005), but were unchanged (all P > 0.6) in the non-immobilized leg. Immobilization induced a 27% decline in the rate of post-absorptive MPS (immobilized, 0.027 +/- 0.003: non-immobilized, 0.037 +/- 0.003% h(-1); P < 0.001). Regardless of dose, AA infusion stimulated a greater rise in MPS in the non-immobilized legs; at 4 h MPS was greater by +54 +/- 12% with low dose and +68 +/- 17% with high dose AA infusion (both P < 0.001). There was some evidence of delayed responsiveness of phosphorylation of Akt to high doses of AA and p70S6k at both doses but no marked differences in that of mTOR, GSK3beta or eEF2. Phosphorylation of focal adhesion kinase (Tyr(576/577)) was reduced (P < 0.05) with immobilization. We observed no change in polyubiquitinated protein content after immobilization. We confirm that 14 days of immobilization reduces MPS in the post-absorptive state and this diminution is reduced but not abolished by increased provision of AA, even at high rates. The immobilization-induced decline in post-absorptive MPS with the 'anabolic resistance' to amino acids can account for much of immobilization-induced muscle atrophy.

Decrease in Akt/PKB signalling in human skeletal muscle by resistance exercise.

We analysed the effects of resistance exercise upon the phosphorylation state of proteins associated with adaptive processes from the Akt/PKB (protein kinase B) and the mitogen-activated protein kinase (MAPK) pathways. Nine healthy young men (21.7 +/- 0.55 year) performed 10 sets of 10 leg extensions at 80% of their 1-RM (repetition maximum). Muscle biopsies were taken from the vastus lateralis at rest, within the first 30 s after exercise and at 24 h post-exercise. Immediately post exercise, the phosphorylation states of Akt/PKB on Thr308 and Ser473 and 4E-BP1 on Thr37/46 (eukaryotic initiation factor 4E-binding protein 1) were decreased (-60 to -90%, P < 0.05). Conversely, the phosphorylation of p70(s6k) (p70 ribosomal S6 kinase) on Thr421/Ser424 was increased more than 20-fold (P < 0.05), and this was associated with a 10- to 50-fold increase in the phosphorylation of p38 and ERK1/2 (extracellular signal-regulated kinase) (P < 0.05). Twenty-four hours post-exercise the phosphorylation state of Akt/PKB on Thr308 was depressed, whereas the phosphorylation of p70(s6k) on Thr421/Ser424 and sarcoplasmic ERK1/2 were elevated. The present results indicate that high-intensity resistance exercise in the fasted state inhibits Akt/PKB and 4E-BP1 whilst concomitantly augmenting MAPK signalling and p70(s6k) on Thr421/Ser424.

Differences in muscle protein synthesis and anabolic signaling in the postabsorptive state and in response to food in 65-80 year old men and women.

Women have less muscle than men but lose it more slowly during aging. To discover potential underlying mechanism(s) for this we evaluated the muscle protein synthesis process in postabsorptive conditions and during feeding in twenty-nine 65-80 year old men (n = 13) and women (n = 16). We discovered that the basal concentration of phosphorylated eEF2(Thr56) was approximately 40% less (P<0.05) and the basal rate of MPS was approximately 30% greater (P = 0.02) in women than in men; the basal concentrations of muscle phosphorylated Akt(Thr308), p70s6k(Thr389), eIF4E(Ser209), and eIF4E-BP1(Thr37/46) were not different between the sexes. Feeding increased (P<0.05) Akt(Thr308) and p70s6k(Thr389) phosphorylation to the same extent in men and women but increased (P<0.05) the phosphorylation of eIF4E(Ser209) and eIF4E-BP1(Thr37/46) in men only. Accordingly, feeding increased MPS in men (P<0.01) but not in women. The postabsorptive muscle mRNA concentrations for myoD and myostatin were not different between sexes; feeding doubled myoD mRNA (P<0.05) and halved that of myostatin (P<0.05) in both sexes. Thus, there is sexual dimorphism in MPS and its control in older adults; a greater basal rate of MPS, operating over most of the day may partially explain the slower loss of muscle in older women.

Effects of resistance exercise with and without creatine supplementation on gene expression and cell signaling in human skeletal muscle.

To test the hypothesis that creatine supplementation would enhance the anabolic responses of muscle cell signaling and gene expression to exercise, we studied nine subjects who received either creatine or a placebo (maltodextrin) for 5 days in a double-blind fashion before undergoing muscle biopsies: at rest, immediately after exercise (10 x 10 repetitions of one-leg extension at 80% 1 repetition maximum), and 24 and 72 h later (all in the morning after fasting overnight). Creatine supplementation decreased the phosphorylation state of protein kinase B (PKB) on Thr308 at rest by 60% (P < 0.05) and that of eukaryotic initiation factor 4E-binding protein on Thr37/46 (4E-BP1) by 30% 24 h postexercise (P < 0.05). Creatine increased mRNA for collagen 1 (alpha(1)), glucose transporter-4 (GLUT-4), and myosin heavy chain I at rest by 250%, 45%, and 80%, respectively, and myosin heavy chain IIA (MHCIIA) mRNA immediately after exercise by 70% (all P < 0.05). Immediately after exercise, and independent of creatine, mRNA for muscle atrophy F-box (MAFbx), MHCIIA, peroxisome proliferator-activated receptor gamma coactivator-1alpha, and interleukin-6 were upregulated (60-350%; P < 0.05); the phosphorylation state of p38 both in the sarcoplasm and nucleus were increased (12- and 25-fold, respectively; both P < 0.05). Concurrently, the phosphorylation states of PKB (Thr308) and 4E-BP1 (Thr37/46) were decreased by 50% and 75%, respectively (P < 0.05). Twenty-four hours postexercise, MAFbx, myostatin, and GLUT-4 mRNA expression decreased below preexercise values (-35 to -50%; P < 0.05); calpain 1 mRNA increased 70% 72 h postexercise (P < 0.05) and at no other time. In conclusion, 5 days of creatine supplementation do not enhance anabolic signaling but increase the expression of certain targeted genes.

The temporal responses of protein synthesis, gene expression and cell signalling in human quadriceps muscle and patellar tendon to disuse.

We hypothesized that rates of myofibrillar and patellar tendon collagen synthesis would fall over time during disuse, the changes being accompanied in muscle by decreases in focal adhesion kinase (FAK) phosphorylation and in gene expression for proteolytic enzymes. We studied nine men (22 +/- 4 years, BMI 24 +/- 3 kg m(-2) (means +/- s.d.) who underwent unilateral lower leg suspension for 23 days; five were studied between 0 and 10 days and four between 10 and 21 days. Muscle and tendon biopsies were taken in the postabsorptive state at days 0, 10 and 21 for measurement of protein synthesis, gene expression and protein phosphorylation. Muscle cross-sectional area decreased by 5.2% at 14 days and 10.0% (both P < 0.001), at 23 days, i.e. 0.5% day(-1), whereas tendon dimensions were constant. Rates of myofibrillar protein synthesis fell (P < 0.01) from 0.047% h(-1) at day 0 to 0.022% h(-1) at 10 days without further changes. Tendon collagen synthetic rates also fell (P < 0.01), from 0.052 to 0.023% h(-1) at 10 days and then to 0.010% h(-1) at 21 days. FAK phosphorylation decreased 30% (P < 0.01) at 10 days. No changes occurred in the amounts/phosphorylation of PKB-P70s6k-mTOR pathway components. Expression of mRNA for MuRF-1 increased approximately 3-fold at 10 days without changes in MAFbx or tripeptidyl peptidase II mRNA, but all decreased between 10 and 21 days. Thus, both myofibrillar and tendon protein synthetic rates show progressive decreases during 21 days of disuse; in muscle, this is accompanied by decreased phosphorylation of FAK, with no marked increases in genes for proteolytic enzymes.

Smoking impairs muscle protein synthesis and increases the expression of myostatin and MAFbx in muscle.

Smoking causes multiple organ dysfunction. The effect of smoking on skeletal muscle protein metabolism is unknown. We hypothesized that the rate of skeletal muscle protein synthesis is depressed in smokers compared with non-smokers. We studied eight smokers (> or =20 cigarettes/day for > or =20 years) and eight non-smokers matched for sex (4 men and 4 women per group), age (65 +/- 3 and 63 +/- 3 yr, respectively; means +/- SEM) and body mass index (25.9 +/- 0.9 and 25.1 +/- 1.2 kg/m(2), respectively). Each subject underwent an intravenous infusion of stable isotope-labeled leucine in conjunction with blood and muscle tissue sampling to measure the mixed muscle protein fractional synthesis rate (FSR) and whole body leucine rate of appearance (Ra) in plasma (an index of whole body proteolysis), the expression of genes involved in the regulation of muscle mass (myostatin, a muscle growth inhibitor, and MAFBx and MuRF-1, which encode E3 ubiquitin ligases in the proteasome proteolytic pathway) and that for the inflammatory cytokine TNF-alpha in muscle, and the concentration of inflammatory markers in plasma (C-reactive protein, TNF-alpha, interleukin-6) which are associated with muscle wasting in other conditions. There were no differences between nonsmokers and smokers in plasma leucine concentration, leucine rate of appearance, and plasma concentrations of inflammatory markers, or TNF-alpha mRNA in muscle, but muscle protein FSR was much less (0.037 +/- 0.005 vs. 0.059 +/- 0.005%/h, respectively, P = 0.004), and myostatin and MAFBx (but not MuRF-1) expression were much greater (by approximately 33 and 45%, respectivley, P < 0.05) in the muscle of smokers than of nonsmokers. We conclude that smoking impairs the muscle protein synthesis process and increases the expression of genes associated with impaired muscle maintenance; smoking therefore likely increases the risk of sarcopenia.

Gene expression responses over 24 h to lengthening and shortening contractions in human muscle: major changes in CSRP3, MUSTN1, SIX1, and FBXO32.

Resistance training using lengthening (eccentric) contractions induces greater increases in muscle size than shortening (concentric) contractions, but the underlying molecular mechanisms are not clear. Using temporal expression profiling, we compared changes in gene expression within 24 h of an acute bout of each type of contractions conducted simultaneously in the quadriceps of different legs. Five healthy young men performed shortening contractions with one leg while the contralateral leg performed lengthening contractions. Biopsies were taken from both legs before exercise and 3, 6, and 24 h afterwards, in the fed state. Expression profiling (n = 3) was performed using a custom-made Affymetrix MuscleChip containing probe sets of approximately 3,300 known genes and expressed sequence tags expressed in skeletal muscle. We identified 51 transcripts differentially regulated between the two exercise modes. Using unsupervised hierarchical clustering, we identified four distinct clusters, three of which corresponded to unique functional categories (protein synthesis, stress response/early growth, and sarcolemmal structure). Using quantitative RT-PCR (n = 5), we verified expression changes (lengthening/shortening) in SIX1 (3 h, -1.9-fold, P < 0.001), CSRP3 (6 h, 2.9-fold, P < 0.05), and MUSTN1 (24 h, 4.3-fold, P < 0.05). We examined whether FBXO32/atrogin-1/MAFbx, a known regulator of protein breakdown and of muscle atrophy was differentially expressed: the gene was downregulated after lengthening contractions (3 h, 2.7-fold, P < 0.05; 6 h, 3.3-fold, P < 0.05; 24 h, 2.3-fold, P < 0.05). The results suggested that lengthening and shortening contractions activated distinct molecular pathways as early as 3 h postexercise. The molecular differences might contribute to mechanisms underlying the physiological adaptations seen with training using the two modes of exercise.

5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside acutely stimulates skeletal muscle 2-deoxyglucose uptake in healthy men.

Activation of AMP-activated protein kinase (AMPK) in rodent muscle by exercise, metformin, 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR), and adiponectin increases glucose uptake. The aim of this study was to determine whether AICAR stimulates muscle glucose uptake in humans. We studied 29 healthy men (aged 26 +/- 8 years, BMI 25 +/- 4 kg/m(2) [mean +/- SD]). Rates of muscle 2-deoxyglucose (2DG) uptake were determined by measuring accumulation of total muscle 2DG (2DG and 2DG-6-phosphate) during a primed, continuous 2DG infusion. The effects of AICAR and exercise on muscle AMPK activity/phosphorylation and 2DG uptake were determined. Whole-body glucose disposal was compared before and during AICAR with the euglycemic-hyperinsulinemic clamp. Muscle 2DG uptake was linear over 9 h (R(2) = 0.88 +/- 0.09). After 3 h, 2DG uptake increased 2.1 +/- 0.8- and 4.7 +/- 1.7-fold in response to AICAR or bicycle exercise, respectively. AMPK alpha(1) and alpha(2) activity or AMPK phosphorylation was unchanged after 20 min or 3 h of AICAR, but AMPK phosphorylation significantly increased immediately and 3 h after bicycle exercise. AICAR significantly increased phosphorylation of extracellular signal-regulated kinase 1/2, but phosphorylation of beta-acetyl-CoA carboxylase, glycogen synthase, and protein kinase B or insulin receptor substrate-1 level was unchanged. Mean whole-body glucose disposal increased by 7% with AICAR from 9.3 +/- 0.6 to 10 +/- 0.6 mg x kg(-1) x min(-1) (P < 0.05). In healthy people, AICAR acutely stimulates muscle 2DG uptake with a minor effect on whole-body glucose disposal.

Tendon collagen synthesis at rest and after exercise in women.

In general, there is a higher incidence of musculoskeletal injuries during physical activity in women than in men. We hypothesized that in women rates of tendon collagen synthesis would be lower than in men at rest and after exercise, especially in the later luteal phase when estrogen and progesterone concentrations are higher than the early follicular phase. We studied tendon collagen fractional synthesis rate (FSR) in 15 young, healthy female subjects in either the early follicular (n = 8) or the late luteal phase (n = 7) 72 h after an acute bout of one-legged exercise (60 min kicking at 67% workload maximum) (72 h) and compared the results with those previously obtained for men. Samples were taken from the patellar tendon in both the exercised and rested legs to determine collagen FSR by the incorporation of [15N]proline into tendon collagen hydroxyproline. There was no effect of menstrual phase on tendon collagen synthesis either at rest or after exercise. However, there was a significant difference between women and men at rest (women = 0.025 +/- 0.002%/h, men = 0.045 +/- 0.008%/h; P < 0.05) and 72 h after exercise (women = 0.027 +/- 0.005%/h; men = 0.058 +/- 0.008%/h). Furthermore, rest and 72-h tendon collagen synthesis were not different in women, whereas in men tendon collagen synthesis remained significantly elevated 72 h after exercise. It is concluded that both in the resting state and after exercise, tendon collagen FSR is lower in women than in men, which may contribute to a lower rate of tissue repair after exercise.

Dietary fiber decreases colonic epithelial cell proliferation and protein synthetic rates in human subjects.

Although it has been proposed that high fiber consumption can prevent proliferative diseases of the colon, the clinical data to support this hypothesis have been inconsistent. To provide a more robust measure of the effects of fiber on colonic mucosal growth than previous studies, we evaluated both cell proliferation and colonic mucosal protein synthesis in nine healthy volunteers after they consumed a typical Western diet (<20 g fiber/day) or a Western diet supplemented with wheat bran (24 g/day) in a randomized crossover design. Biopsies taken from the sigmoid colon were used to assess mucosal proliferation by determining proliferating cell nuclear antigen (PCNA) in crypt cells and to assess mucosal protein synthetic rate using stable isotopically labeled leucine infusion. Fiber supplementation produced a 12% decrease in labeling index (%crypt cells stained with PCNA) (P < 0.001) and an 11% decrease in mucosal protein fractional synthetic rate (FSR; P < 0.05). Moreover, mucosal protein FSR correlated directly with labeling index (r2= 0.22, P < 0.05). These data demonstrate that increased wheat bran consumption decreases colonic mucosal proliferation and support the potential importance of dietary fiber in preventing proliferative diseases of the colon.