Rebuttal from Marlou L. Dirks, Benjamin T. Wall and Francis B. Stephens

This is the final version. Available on open access from Wiley via the DOI in this recordThis article is part of a CrossTalk debate. Click the links to read the other articles in this debate: https://doi.org/10.1113/JP278219, https://doi.org/10.1113/JP279714, https://doi.org/10.1113/JP27822

A single day of bed rest, irrespective of energy balance, does not affect skeletal muscle gene expression or insulin sensitivity

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this record.The initial metabolic and molecular events that underpin disuse-induced skeletal muscle deconditioning, and the contribution of energy balance, remain to be investigated. Ten young, healthy males (age: 25 ± 1 y; BMI: 25.3 ± 0.8 kg m-2 ) underwent three 24 h laboratory-based experimental periods in a randomized, crossover manner: 1) controlled habitual physical activity with an energy-balanced diet (CON); 2) strict bed rest with a diet to maintain energy balance (BR-B); and 3) strict bed rest with a diet identical to CON, consequently resulting in positive energy balance. Continuous glucose monitoring was performed throughout each visit, with vastus lateralis muscle biopsies and an oral glucose tolerance test performed before and after. In parallel with muscle samples collected from a previous 7-day bed rest study, biopsies were used to examine expression of genes associated with the regulation of muscle mass and insulin sensitivity. A single day of bed rest, irrespective of energy balance, did not lead to overt changes in whole-body substrate oxidation, indices of insulin sensitivity (i.e. HOMA-IR (BR-B: from 2.7 ± 1.7 to 3.1 ± 1.5, P > 0.05), Matsuda (BR-B: from 5.9 ± 3.3 to 5.2 ± 2.9, P > 0.05)), or 24 h glycaemic control/variability compared to CON. Seven days of bed rest led to ∼30-55% lower expression of genes involved in insulin signalling, lipid storage/oxidation, and muscle protein breakdown, whereas no such changes were observed after one day of bed rest. In conclusion, more than one day of physical inactivity is required to observe the insulin resistance and robust skeletal muscle transcriptional responses associated with bed rest and consequent alterations in energy balance.BTW received internal funding from the College of Life and Environmental Sciences, University of Exeter, to support this project. None of the other authors received funding from any funding agency in the public, commercial or not-for-profit sectors to conduct this research

Comparing the frequency of common genetic variants and haplotypes between carriers and non-carriers of BRCA1 and BRCA2 deleterious mutations in Australian women diagnosed with breast cancer before 40 years of age

BACKGROUND: BRCA1 and BRCA2 mutations are found in a proportion of families with multiple early-onset breast cancers. There are a large number of different deleterious mutations in both genes, none of which would be detectable using standard genetic association studies. Single common variants and haplotypes of common variants may capture groups of deleterious mutations since some low prevalence haplotypes of common variants occur more frequently among chromosomes that carry rare, deleterious mutations than chromosomes that do not. METHODS: DNA sequence data for BRCA1 and BRCA2 was obtained from 571 participants from the Australian Breast Cancer Family Study. Genetic variants were classified as either deleterious mutations or common genetic variants. Variants tagging common polymorphisms were selected and haplotypes resolved using Haploview. Their frequency was compared to those with and without deleterious mutations using a permutation test. RESULTS: A common genetic variant in BRCA1 (3232A > G) was found to be over-represented in deleterious mutation carriers (p = 0.05), whereas a common genetic variant in BRCA2 (1342A > C) occurred less frequently in deleterious mutation carriers (p = 0.04). All four of the common BRCA1 variants used to form haplotypes occurred more frequently in the deleterious mutation carriers when compared to the non-carriers, but there was no evidence of a difference in the distributions between the two groups (p = 0.34). In BRCA2, all four common variants were found to occur less frequently in the deleterious mutation carriers when compared to non-carriers, but the evidence for difference in the distribution between the two groups was weak (p = 0.16). Several less common haplotypes of common BRCA1 variants were found to be over-represented among deleterious mutation carriers but there was no evidence for this at the population level. In BRCA2, only the most common haplotype was found to occur more frequently in deleterious mutation carriers, with again no evidence at the population level. CONCLUSIONS: We observed differences in the frequency of common genetic variants of the BRCA1 and BRCA2 and their haplotypes between early-onset breast cancer cases who did and did not carry deleterious mutations in these genes. Although our data provide only weak evidence for a difference in frequencies at the population level, the number of deleterious mutation carriers was low and the results may yet be substantiated in a larger study using pooled data

Short-term disuse does not affect postabsorptive or postprandial muscle protein fractional breakdown rates

This is the final version. Available on open access from Wiley via the DOI in this recordBACKGROUND: The decline in postabsorptive and postprandial muscle protein fractional synthesis rates (FSR) does not quantitatively account for muscle atrophy during uncomplicated, short-term disuse, when atrophy rates are the highest. We sought to determine whether 2 days of unilateral knee immobilization affects mixed muscle protein fractional breakdown rates (FBR) during postabsorptive and simulated postprandial conditions. METHODS: Twenty-three healthy, male participants (age: 22 ± 1 year; height: 179 ± 1 cm; body mass: 73.4 ± 1.5 kg; body mass index 22.8 ± 0.5 kg·m-2 ) took part in this randomized, controlled study. After 48 h of unilateral knee immobilization, primed continuous intravenous l-[15 N]-phenylalanine and l-[ring-2 H5 ]-phenylalanine infusions were used for parallel determinations of FBR and FSR, respectively, in a postabsorptive (saline infusion; FAST) or simulated postprandial state (67.5 mg·kg body mass-1 ·h-1 amino acid infusion; FED). Bilateral m. vastus lateralis biopsies from the control (CON) and immobilized (IMM) legs, and arterialized-venous blood samples, were collected throughout. RESULTS: Amino acid infusion rapidly increased plasma phenylalanine (59 ± 9%), leucine (76 ± 5%), isoleucine (109 ± 7%) and valine (42 ± 4%) concentrations in FED only (all P  0.05). However, immobilization decreased FSR (P < 0.05) in both FAST (0.071 ± 0.004 vs. 0.086 ± 0.007%·h-1 ; IMM vs CON, respectively) and FED (0.066 ± 0.016 vs. 0.119 ± 0.016%·h-1 ; IMM vs CON, respectively). Consequently, immobilization decreased net muscle protein balance (P < 0.05) and to a greater extent in FED (CON: -0.012 ± 0.025; IMM: -0.095 ± 0.023%·h-1 ; P < 0.05) than FAST (CON: -0.064 ± 0.020; IMM: -0.072 ± 0.017%·h-1 ). CONCLUSIONS: We conclude that merely 2 days of leg immobilization does not modulate postabsorptive and simulated postprandial muscle protein breakdown rates. Instead, under these conditions the muscle negative muscle protein balance associated with brief periods of experimental disuse is driven near exclusively by reduced basal muscle protein synthesis rates and anabolic resistance to amino acid administration.Nutricia Research FoundationUniversity of ExeterBeachbody LLCNational Institute of Agin

Daily mycoprotein consumption for one week does not affect insulin sensitivity or glycaemic control but modulates the plasma lipidome in healthy adults: a randomised controlled trial

This is the author accepted manuscript. The final version is available from Cambridge University Press via the DOI in this record.Mycoprotein consumption has been shown to improve acute postprandial glycaemic control and decrease circulating cholesterol concentrations. We investigated the impact of incorporating mycoprotein into the diet on insulin sensitivity (IS), glycaemic control and plasma lipoprotein composition. Twenty healthy adults participated in a randomised, parallel-group trial in which they consumed a 7 d fully-controlled diet where lunch and dinner contained either meat/fish (CON) or mycoprotein (MYC) as the primary source of dietary protein. Oral glucose tolerance tests were performed pre- and post- intervention, and 24h continuous blood glucose monitoring was applied throughout. Fasting plasma samples were obtained pre- and post- intervention and were analysed using quantitative, targeted NMR-based metabonomics. There were no changes within or between groups in blood glucose or serum insulin responses, nor in IS (Cederholm; 51±3 to 51±3 and 54±3 to 53±3 mg.L2/mmol.mU.min in CON and MYC, respectively; P<0.05) or 24 h glycaemic profiles. No differences between groups were found for 171 of the 224 metabonomic targets. Forty five lipid concentrations of different lipoprotein fractions (VLDL, LDL, IDL and HDL) remained unchanged in CON but showed a coordinated decrease (7-27 %; all P<0.05) in MYC. Total plasma cholesterol, free-C, LDL-C, HDL2-C, DHA and omega-3 fatty acids decreased to a larger degree in MYC (14-19 %) compared with CON (3-11 %; P<0.05). Substituting meat/fish for mycoprotein twice-daily for one week did not modulate whole-body IS or glycaemic control but resulted in changes to plasma lipid composition; the latter primarily consisting of a coordinated reduction in circulating cholesterol containing lipoproteins.QuornMarlow Foods Lt

High-fat overfeeding does not exacerbate rapid changes in forearm glucose and fatty acid balance during immobilization

This is the author accepted manuscript. The final version is available from Oxford University Press via the DOI in this recordCONTEXT: Physical inactivity and high-fat overfeeding have been shown to independently induce insulin resistance. OBJECTIVE: Establish the contribution of muscle disuse and lipid availability to the development of inactivity-induced insulin resistance. Design, setting, participants, and interventions: Twenty healthy males underwent seven days of forearm cast immobilization combined with a fully-controlled eucaloric (CON, n=10, age 23±2 yr, BMI 23.8±1.0 kg·m-2) or high-fat diet providing 50% excess energy from fat (HFD, n=10, age 23±2 yr, BMI 22.4±0.8 kg·m-2). MAIN OUTCOME MEASURES: Prior to casting, and following 2 and 7 days of immobilization, forearm glucose uptake (FGU) and non-esterified fatty acid (NEFA) balance were assessed using the arterialized venous-deep venous (AV-V) forearm balance method following ingestion of a mixed macronutrient drink. RESULTS: Seven days of HFD increased body weight by 0.9±0.2 kg (P=0.002), but did not alter fasting, arterialized whole-blood glucose and serum insulin concentrations or the associated HOMA-IR or Matsuda indices. Two and seven days of forearm immobilization led to a 40±7% and 52±7% decrease in FGU, respectively (P<0.001), with no difference between day 2 and 7 and no effect of HFD. Forearm NEFA balance tended to increase following two and seven days of immobilization (P=0.095). CONCLUSIONS: forearm immobilization leads to a rapid and substantial decrease in FGU, which is accompanied by an increase in forearm NEFA balance but is not exacerbated by excess dietary fat intake. Altogether, our data suggest that disuse-induced insulin resistance of glucose metabolism is occurs as a physiological adaptation in response to the removal of muscle contraction.Physiological Societ

Viral population estimation using pyrosequencing

The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an EM algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.Comment: 23 pages, 13 figure

The impact of forearm immobilization and acipimox administration on muscle amino acid metabolism and insulin sensitivity in healthy, young volunteers

This is the author accepted manuscript. The final version is available on open access from the American Physiological Society via the DOI in this recordAlthough the mechanisms underpinning short-term muscle disuse atrophy and associated insulin resistance remain to be elucidated, perturbed lipid metabolism might be involved. Our aim was to determine the impact of acipimox administration (i.e. pharmacologically lowering circulating non-esterified fatty acid (NEFA) availability) on muscle amino acid metabolism and insulin sensitivity during short-term disuse. Eighteen healthy individuals (age 22±1 years, BMI 24.0±0.6 kg·m-2) underwent 2 days forearm immobilization with placebo (PLA; n=9) or acipimox (ACI; 250 mg Olbetam; n=9) ingestion four times daily. Before and after immobilization, whole-body glucose disposal rate (GDR), forearm glucose uptake (FGU, i.e. muscle insulin sensitivity), and amino acid kinetics were measured under fasting and hyperinsulinaemic-hyperaminoacidaemic-euglycaemic clamp conditions using forearm balance and L-[ring-2H5]-phenylalanine infusions. Immobilization did not affect GDR but decreased insulin-stimulated FGU in both groups; more so in ACI (from 53±8 to 12±5 µmol·min-1) than PLA (from 52±8 to 38±13 µmol·min-1; P<0.05). In ACI only, and in contrast to our hypothesis, fasting arterialised NEFA concentrations were elevated to 1.3±0.1 mmol·L-1 post-immobilization (P<0.05), and fasting forearm NEFA balance increased ~4-fold (P=0.10). Forearm phenylalanine net balance decreased following immobilization (P<0.10), driven by increased Ra (from 32±5 (fasting) and 21±4 (clamp) pre-immobilization to 53±8 and 31±4 post-immobilization; P<0.05) while Rd was unaffected by disuse or acipimox. Disuse-induced insulin resistance is accompanied by early signs of negative net muscle amino acid balance, which is driven by accelerated muscle amino acid efflux. Acutely elevated NEFA availability worsened muscle insulin resistance without affecting amino acid kinetics, suggesting increased muscle NEFA uptake may contribute to inactivity-induced insulin resistance but does not cause anabolic resistance.Wellcome TrustNational Institute of Agin

Association of a Bovine Prion Gene Haplotype with Atypical BSE

Background: Atypical bovine spongiform encephalopathies (BSEs) are recently recognized prion diseases of cattle. Atypical BSEs are rare; approximately 30 cases have been identified worldwide. We tested prion gene (PRNP) haplotypes for an association with atypical BSE. Methodology/Principle Findings: Haplotype tagging polymorphisms that characterize PRNP haplotypes from the promoter region through the three prime untranslated region of exon 3 (25.2 kb) were used to determine PRNP haplotypes of six available atypical BSE cases from Canada, France and the United States. One or two copies of a distinct PRNP haplotype were identified in five of the six cases (p = 1.36×10-4, two-tailed Fisher’s exact test; CI95% 0.263–0.901, difference between proportions). The haplotype spans a portion of PRNP that includes part of intron 2, the entire coding region of exon 3 and part of the three prime untranslated region of exon 3 (13 kb). Conclusions/Significance: This result suggests that a genetic determinant in or near PRNP may influence susceptibility of cattle to atypical BSE