Marie C. Limb

Intake of low-dose leucine-rich essential amino acids stimulates muscle anabolism equivalently to bolus whey protein in older women, at rest and after exercise

Dysregulated anabolic responses to nutrition/exercise may contribute to sarcopenia; however, these characteristics are poorly defined in female populations. We determined the effects of two feeding regimes in older women (66 ± 2.5 yr; n = 8/group): bolus whey protein (WP-20 g) or novel low-dose leucine-enriched essential amino acids (EAA) [LEAA; 3 g (40% leucine)]. Using [(13)C6]phenylalanine infusions, we quantified muscle (MPS) and albumin (APS) protein synthesis at baseline and in response to both feeding (FED) and feeding plus exercise (FED-EX; 6 × 8 knee extensions at 75% 1-repetition maximum). We also quantified plasma insulin/AA concentrations, whole leg (LBF)/muscle microvascular blood flow (MBF), and muscle anabolic signaling by phosphoimmunoblotting. Plasma insulinemia and EAA/aemia were markedly greater after WP than LEAA (P < 0.001). Neither LEAA nor WP modified LBF in response to FED or FED-EX, whereas MBF increased to a similar extent in both groups only after FED-EX (P < 0.05). In response to FED, both WP and LEAA equally stimulated MPS 0-2 h (P < 0.05), abating thereafter (0-4 h, P > 0.05). In contrast, after FED-EX, MPS increased at 0-2 h and remained elevated at 0-4 h (P < 0.05) with both WP and LEAA. No anabolic signals quantifiably increased after FED, but p70 S6K1 Thr(389) increased after FED-EX (2 h, P < 0.05). APS increased similarly after WP and LEAA. Older women remain subtly responsive to nutrition ± exercise. Intriguingly though, bolus WP offers no trophic advantage over LEAA.

Toxigenic Helicobacter pylori Infection Precedes Gastric Hypochlorhydria in Cancer Relatives, and H. pylori Virulence Evolves in These Families

Purpose:Helicobacter pylori infection by virulent strains is associated with gastric adenocarcinoma. We aimed to determine whether infection with virulent H. pylori preceeded precancerous gastric hypochlorhydria and atrophy in gastric cancer relatives and quantify the extent of virulence factor evolution. Experimental Design:H. pylori strains from 51 Scottish gastric cancer relatives were characterized by genetic fingerprinting and typing the vacuolating cytotoxin gene (vacA), the cytotoxin-associated gene (cagA), and housekeeping genes. We phenotyped strains by coculture with gastric epithelial cells and assessing vacuolation (microscopy), CagA tyrosine phosphorylation (immunoblot), and interleukin-8 secretion (ELISA). Results: Toxigenic (vacA type s1/m1) H. pylori was associated with precancerous gastric hypochlorhydria (P < 0.01). Adult family members with this type of H. pylori had the same strain as currently noncohabiting adult family members in 68% cases, implying acquisition during childhood from each other or a common source. We analyzed different isolates of the same strain within families and showed that H. pylori commonly microevolved to change virulence: this occurred in 22% individuals and a striking 44% cases where the strain was shared within families. Microevolution in vacA occurred by extragenomic recombination and in cagA by this or duplication/deletion. Microevolution led to phenotypic changes in virulence. Passage of microevolved strains could be tracked within families. Conclusions: Toxigenic H. pylori infection precedes and so likely causes gastric hypochlorhydria, suggesting that virulent H. pylori increases cancer risk by causing this condition. Microevolution of virulence genes is common within families of gastric cancer patients and changes H. pylori virulence.

Naloxone as part of a prolonged release oxycodone/naloxone combination reduces oxycodone-induced slowing of gastrointestinal transit in healthy volunteers

Objectives: This exploratory study in healthy volunteers investigated the effect of single doses of oxycodone on gastrointestinal (GI) transit time and the degree to which a single dose of naloxone reverses the oxycodone-induced effect. Methods: Fifteen healthy male volunteers received: oxycodone 10 and 20 mg, oxycodone/naloxone 10/5 and 20/10 mg (all as prolonged release tablets) and placebo. Each dose was radiolabelled and administered with a capsule containing radiolabelled resin (surrogate for GI contents). Results: Scintigraphic analysis showed that 20 mg oxycodone significantly increased colon arrival time (mean 7.19 vs 5.15 h for placebo, p = 0.0159). Mean colon arrival time for oxycodone/naloxone 20/10 mg (5.16 h) was similar to placebo, although the difference between oxycodone/naloxone 20/10 mg versus oxycodone 20 mg was not significant (p = 0.0653). Colonic geometric centre analysis showed a significant increase in mean time for the resin to reach the colon following oxycodone 10 and 20 mg compared with placebo (increases of 5.3 and 8.8 h). There was no significant effect of naloxone at the lower dose; however, oxycodone/naloxone 20/10 mg significantly reduced mean colonic transit time by 2.1 h (p = 0.0376). Conclusion: A single dose of oxycodone 20 mg significantly prolonged GI transit time but this effect was reduced by co-administration of naloxone.

Fuel for the work required: a practical approach to amalgamating train-low paradigms for endurance athletes

Using an amalgamation of previously studied “train‐low” paradigms, we tested the effects of reduced carbohydrate (CHO) but high leucine availability on cell‐signaling responses associated with exercise‐induced regulation of mitochondrial biogenesis and muscle protein synthesis (MPS). In a repeated‐measures crossover design, 11 males completed an exhaustive cycling protocol with high CHO availability before, during, and after exercise (HIGH) or alternatively, low CHO but high protein (leucine enriched) availability (LOW + LEU). Muscle glycogen was different (P < 0.05) pre‐exercise (HIGH: 583 ± 158, LOW + LEU: 271 ± 85 mmol kg−1 dw) but decreased (P < 0.05) to comparable levels at exhaustion (≈100 mmol kg−1 dw). Despite differences (P < 0.05) in exercise capacity (HIGH: 158 ± 29, LOW + LEU: 100 ± 17 min), exercise induced (P < 0.05) comparable AMPKα2 (3–4‐fold) activity, PGC‐1α (13‐fold), p53 (2‐fold), Tfam (1.5‐fold), SIRT1 (1.5‐fold), Atrogin 1 (2‐fold), and MuRF1 (5‐fold) gene expression at 3 h post‐exercise. Exhaustive exercise suppressed p70S6K activity to comparable levels immediately post‐exercise (≈20 fmol min−1 mg−1). Despite elevated leucine availability post‐exercise, p70S6K activity remained suppressed (P < 0.05) 3 h post‐exercise in LOW + LEU (28 ± 14 fmol min−1 mg−1), whereas muscle glycogen resynthesis (40 mmol kg−1 dw h−1) was associated with elevated (P < 0.05) p70S6K activity in HIGH (53 ± 30 fmol min−1 mg−1). We conclude: (1) CHO restriction before and during exercise induces “work‐efficient” mitochondrial‐related cell signaling but; (2) post‐exercise CHO and energy restriction maintains p70S6K activity at basal levels despite feeding leucine‐enriched protein. Our data support the practical concept of “fuelling for the work required” as a potential strategy for which to amalgamate train‐low paradigms into periodized training programs.

The effects of resistance exercise training on macro‐ and micro‐circulatory responses to feeding and skeletal muscle protein anabolism in older men

Increases in limb blood flow in response to nutrition are reduced in older age. Muscle microvascular blood flow (MBF) in response to nutrition is also reduced with advancing age and this may contribute to age‐related ‘anabolic resistance’. Resistance exercise training (RET) can rejuvenate limb blood flow responses to nutrition in older individuals. We report here that 20 weeks of RET also restores muscle MBF in older individuals. Restoration of MBF does not, however, enhance muscle anabolic responses to nutrition.

Pharmacological enhancement of leg and muscle microvascular blood flow does not augment anabolic responses in skeletal muscle of young men under fed conditions

Skeletal muscle anabolism associated with postprandial plasma aminoacidemia and insulinemia is contingent upon amino acids (AA) and insulin crossing the microcirculation-myocyte interface. In this study, we hypothesized that increasing muscle microvascular blood volume (flow) would enhance fed-state anabolic responses in muscle protein turnover. We studied 10 young men (23.2 ± 2.1 yr) under postabsorptive and fed [iv Glamin (∼10 g AA), glucose ∼7.5 mmol/l] conditions. Methacholine was infused into the femoral artery of one leg to determine, via bilateral comparison, the effects of feeding alone vs. feeding plus pharmacological vasodilation. We measured leg blood flow (LBF; femoral artery) by Doppler ultrasound, muscle microvascular blood volume (MBV) by contrast-enhanced ultrasound (CEUS), muscle protein synthesis (MPS) and breakdown (MPB; a-v balance modeling), and net protein balance (NPB) using [1,2-(13)C2]leucine and [(2)H5]phenylalanine tracers via gas chromatography-mass spectrometry (GC-MS). Indexes of anabolic signaling/endothelial activation (e.g., Akt/mTORC1/NOS) were assessed using immunoblotting techniques. Under fed conditions, LBF (+12 ± 5%, P < 0.05), MBV (+25 ± 10%, P < 0.05), and MPS (+129 ± 33%, P < 0.05) increased. Infusion of methacholine further enhanced LBF (+126 ± 12%, P < 0.05) and MBV (+79 ± 30%, P < 0.05). Despite these radically different blood flow conditions, neither increases in MPS in response to feeding (0.04 ± 0.004 vs. 0.08 ± 0.01%/h, P < 0.05) nor improvements in NPB (-4.4 ± 2.4 vs. 16.4 ± 5.7 nmol Phe·100 ml leg(-1)·min(-1), P < 0.05) were affected by methacholine infusion (MPS 0.07 ± 0.01%/h; NPB 24.0 ± 7.7 nmol Phe·100 ml leg(-1)·min(-1)), whereas MPB was unaltered by either feeding or infusion of methacholine. Thus, enhancing LBF/MBV above that occurring naturally with feeding alone does not improve muscle anabolism.

Effects of leucine-enriched essential amino acid and whey protein bolus dosing upon skeletal muscle protein synthesis at rest and after exercise in older women

Summary Background & aims Impaired anabolic responses to nutrition and exercise contribute to loss of skeletal muscle mass with ageing (sarcopenia). Here, we tested responses of muscle protein synthesis (MPS), in the under represented group of older women, to leucine-enriched essential amino acids (EAA) in comparison to a large bolus of whey protein (WP). Methods Twenty-four older women (65 ± 1 y) received (N = 8/group) 1.5 g leucine-enriched EAA supplements (LEAA_1.5), 6 g LEAA (LEAA_6) in comparison to 40 g WP. A primed constant I.V infusion of 13C6-phenylalanine was used to determine MPS at baseline and in response to feeding (FED) and feeding-plus-exercise (FED-EX; 6 × 8 unilateral leg extensions; 75%1-RM). We quantified plasma insulin/AA concentrations, leg femoral blood flow (LBF)/muscle microvascular blood flow (MBF), and anabolic signalling via immunoblotting. Results Plasma insulineamia and EAAemia were greater and more prolonged with WP than LEAA, although LEAA_6 peaked at similar levels to WP. Neither LEAA or WP modified LBF or MBF. FED increased MPS similarly in the LEAA_1.5, LEAA_6 and WP (P < 0.05) groups over 0–2 h, with MPS significantly higher than basal in the LEAA_6 and WP groups only over 0–4 h. However, FED-EX increased MPS similarly across all the groups from 0 to 4 h (P < 0.05). Only p-p70S6K1 increased with WP at 2 h in FED (P < 0.05), and at 2/4 h in FED-EX (P < 0.05). Conclusions In conclusion, LEAA_1.5, despite only providing 0.6 g of leucine, robustly (perhaps maximally) stimulated MPS, with negligible trophic advantage of greater doses of LEAA or even to 40 g WP. Highlighting that composition of EAA, in particular the presence of leucine rather than amount is most crucial for anabolism.

Acute cocoa flavanol supplementation improves muscle macro- and microvascular but not anabolic responses to amino acids in older men

The anabolic effects of nutrition on skeletal muscle may depend on adequate skeletal muscle perfusion, which is impaired in older people. Cocoa flavanols have been shown to improve flow-mediated dilation, an established measure of endothelial function. However, their effect on muscle microvascular blood flow is currently unknown. Therefore, the objective of this study was to explore links between the consumption of cocoa flavanols, muscle microvascular blood flow, and muscle protein synthesis (MPS) in response to nutrition in older men. To achieve this objective, leg blood flow (LBF), muscle microvascular blood volume (MBV), and MPS were measured under postabsorptive and postprandial (intravenous Glamin (Fresenius Kabi, Germany), dextrose to sustain glucose ∼7.5 mmol·L(-1)) conditions in 20 older men. Ten of these men were studied with no cocoa flavanol intervention and a further 10 were studied with the addition of 350 mg of cocoa flavanols at the same time that nutrition began. Leg (femoral artery) blood flow was measured by Doppler ultrasound, muscle MBV by contrast-enhanced ultrasound using Definity (Lantheus Medical Imaging, Mass., USA) perflutren contrast agent and MPS using [1, 2-(13)C2]leucine tracer techniques. Our results show that although older individuals do not show an increase in LBF or MBV in response to feeding, these absent responses are apparent when cocoa flavanols are given acutely with nutrition. However, this restoration in vascular responsiveness is not associated with improved MPS responses to nutrition. We conclude that acute cocoa flavanol supplementation improves muscle macro- and microvascular responses to nutrition, independently of modifying muscle protein anabolism.

Impact of the calcium form of β-hydroxy-β-methylbutyrate upon human skeletal muscle protein metabolism.

Summary Background & aims β-hydroxy-β-methylbutyrate (HMB) is purported as a key nutritional supplement for the preservation of muscle mass in health, disease and as an ergogenic aid in exercise. Of the two available forms of HMB (calcium (Ca-HMB) salt or free acid (FA-HMB)) – differences in plasma bioavailability have been reported. We previously reported that ∼3 g oral FA-HMB increased muscle protein synthesis (MPS) and reduced muscle protein breakdown (MPB). The objective of the present study was to quantify muscle protein metabolism responses to oral Ca-HMB. Methods Eight healthy young males received a primed constant infusion of 1,2 13C2 leucine and 2H5 phenylalanine to assess MPS (by tracer incorporation in myofibrils) and MPB (via arterio-venous (A-V) dilution) at baseline and following provision of ∼3 g of Ca-HMB; muscle anabolic (MPS) and catabolic (MPB) signalling was assessed via immunoblotting. Results Ca-HMB led a significant and rapid (<60 min) peak in plasma HMB concentrations (483.6 ± 14.2 μM, p < 0.0001). This rise in plasma HMB was accompanied by increases in MPS (PA: 0.046 ± 0.004%/h, CaHMB: 0.072 ± 0.004%/h, p < 0001) and suppressions in MPB (PA: 7.6 ± 1.2 μmol Phe per leg min−1, Ca-HMB: 5.2 ± 0.8 μmol Phe per leg min−1, p < 0.01). Increases in the phosphorylation of mTORc1 substrates i.e. p70S6K1 and RPS6 were also observed, with no changes detected in the MPB targets measured. Conclusions These findings support the pro-anabolic properties of HMB via mTORc1, and show that despite proposed differences in bioavailability, Ca-HMB provides a comparable stimulation to MPS and suppression of MPB, to FA-HMB, further supporting its use as a pharmaconutrient in the modulation of muscle mass.