Pyruvate Kinase M2 Supports Muscle Progenitor Cell Proliferation but Is Dispensable for Skeletal Muscle Regeneration after Injury.

BACKGROUND: Skeletal muscle progenitor cells (MPCs) repair damaged muscle postinjury. Pyruvate kinase M2 (PKM2) is a glycolytic enzyme (canonical activity) that can also interact with other proteins (noncanonical activity) to modify diverse cellular processes. Recent evidence links PKM2 to MPC proliferation. OBJECTIVES: This study aimed to understand cellular roles for PKM2 in MPCs and the necessity of PKM2 in MPCs for muscle regeneration postinjury. METHODS: Cultured, proliferating MPCs (C2C12 cells) were treated with a short hairpin RNA targeting PKM2 or small molecules that selectively affect canonical and noncanonical PKM2 activity (shikonin and TEPP-46). Cell number was measured, and RNA-sequencing and metabolic assays were used in follow-up experiments. Immunoprecipitation coupled to proteomics was used to identify binding partners of PKM2. Lastly, an MPC-specific PKM2 knockout mouse was generated and challenged with a muscle injury to determine the impact of PKM2 on regeneration. RESULTS: When the noncanonical activity of PKM2 was blocked or impaired, there was an increase in reactive oxygen species concentrations (1.6-2.0-fold, P < 0.01). Blocking noncanonical PKM2 activity also increased lactate excretion (1.2-1.6-fold, P < 0.05) and suppressed mitochondrial oxygen consumption (1.3-1.6-fold, P < 0.01). Glutamate dehydrogenase 1 (GLUD1) was identified as a PKM2 binding partner and blocking noncanonical PKM2 activity increased GLUD activity (1.5-1.6-fold, P < 0.05). Mice with an MPC-specific PKM2 deletion did not demonstrate impaired muscle regeneration. CONCLUSIONS: The results suggest that the noncanonical activity of PKM2 is important for MPC proliferation in vitro and demonstrate GLUD1 as a PKM2 binding partner. Because no impairments in muscle regeneration were detected in a mouse model, the endogenous environment may compensate for loss of PKM2.

The effect of dairy products and non-dairy snacks on food intake, subjective appetite and cortisol levels in children: a randomized control study.

Dairy snacks are available in various physical forms and their consumption is linked to improved metabolic health. The objective of this study was to determine the effect of dairy snacks of different physical forms on short-term food intake (FI), subjective appetite, and the stress hormone, cortisol, in children. Following a repeated-measures crossover design, 40 children aged 9-14 years randomly consumed 1 of 5 isoenergetic (180 kcal) snacks per study session. These snacks included solid (potato chips, cookies, and cheese), semi-solid (Greek yogurt), and fluid (2% fat milk) snacks. FI was measured 120 min after snack consumption. Subjective appetite was measured at 0 (immediately before the snack), 15, 30, 45, 60, 90, and 120 min. Salivary cortisol (n = 18) was measured after the Greek yogurt and cookie snacks at 0, 30, 60, 90, and 120 min. FI did not differ between snacks (P = 0.15). The Greek yogurt (P < 0.0001) and cheese (P = 0.0009) snacks reduced average appetite compared with the 2% fat milk snack. Salivary cortisol levels were not affected by snack (P = 0.84). This study demonstrates that dairy snacks are as effective as other popular snacks at influencing subsequent FI. However, solid and semi-solid dairy snacks are more effective at repressing subjective appetite than a fluid dairy snack. Registered at ClinicalTrials.gov (NCT02484625). Novelty: Milk, Greek yogurt and cheese have a similar effect on short-term food intake in children as popular potato chips and cookie snacks. Solid, semi-solid and liquid snacks have a similar effect on short-term food intake in children.

Extracellular serine and glycine are required for mouse and human skeletal muscle stem and progenitor cell function.

OBJECTIVE: Skeletal muscle regeneration relies on muscle-specific adult stem cells (MuSCs), MuSC progeny, muscle progenitor cells (MPCs), and a coordinated myogenic program that is influenced by the extracellular environment. Following injury, MPCs undergo a transient and rapid period of population expansion, which is necessary to repair damaged myofibers and restore muscle homeostasis. Certain pathologies (e.g., metabolic diseases and muscle dystrophies) and advanced age are associated with dysregulated muscle regeneration. The availability of serine and glycine, two nutritionally non-essential amino acids, is altered in humans with these pathologies, and these amino acids have been shown to influence the proliferative state of non-muscle cells. Our objective was to determine the role of serine/glycine in MuSC/MPC function. METHODS: Primary human MPCs (hMPCs) were used for in vitro experiments, and young (4-6 mo) and old (>20 mo) mice were used for in vivo experiments. Serine/glycine availability was manipulated using specially formulated media in vitro or dietary restriction in vivo followed by downstream metabolic and cell proliferation analyses. RESULTS: We identified that serine/glycine are essential for hMPC proliferation. Dietary restriction of serine/glycine in a mouse model of skeletal muscle regeneration lowered the abundance of MuSCs 3 days post-injury. Stable isotope-tracing studies showed that hMPCs rely on extracellular serine/glycine for population expansion because they exhibit a limited capacity for de novo serine/glycine biosynthesis. Restriction of serine/glycine to hMPCs resulted in cell cycle arrest in G0/G1. Extracellular serine/glycine was necessary to support glutathione and global protein synthesis in hMPCs. Using an aged mouse model, we found that reduced serine/glycine availability augmented intermyocellular adipocytes 28 days post-injury. CONCLUSIONS: These studies demonstrated that despite an absolute serine/glycine requirement for MuSC/MPC proliferation, de novo synthesis was inadequate to support these demands, making extracellular serine and glycine conditionally essential for efficient skeletal muscle regeneration.

A defined N6-methyladenosine (m6A) profile conferred by METTL3 regulates muscle stem cell/myoblast state transitions.

Muscle-specific adult stem cells (MuSCs) are required for skeletal muscle regeneration. To ensure efficient skeletal muscle regeneration after injury, MuSCs must undergo state transitions as they are activated from quiescence, give rise to a population of proliferating myoblasts, and continue either to terminal differentiation, to repair or replace damaged myofibers, or self-renewal to repopulate the quiescent population. Changes in MuSC/myoblast state are accompanied by dramatic shifts in their transcriptional profile. Previous reports in other adult stem cell systems have identified alterations in the most abundant internal mRNA modification, N6-methyladenosine (m6A), conferred by its active writer, METTL3, to regulate cell state transitions through alterations in the transcriptional profile of these cells. Our objective was to determine if m6A-modification deposition via METTL3 is a regulator of MuSC/myoblast state transitions in vitro and in vivo. Using liquid chromatography/mass spectrometry we identified that global m6A levels increase during the early stages of skeletal muscle regeneration, in vivo, and decline when C2C12 myoblasts transition from proliferation to differentiation, in vitro. Using m6A-specific RNA-sequencing (MeRIP-seq), a distinct profile of m6A-modification was identified, distinguishing proliferating from differentiating C2C12 myoblasts. RNAi studies show that reducing levels of METTL3, the active m6A methyltransferase, reduced global m6A levels and forced C2C12 myoblasts to prematurely differentiate. Reducing levels of METTL3 in primary mouse MuSCs prior to transplantation enhanced their engraftment capacity upon primary transplantation, however their capacity for serial transplantation was lost. In conclusion, METTL3 regulates m6A levels in MuSCs/myoblasts and controls the transition of MuSCs/myoblasts to different cell states. Furthermore, the first transcriptome wide map of m6A-modifications in proliferating and differentiating C2C12 myoblasts is provided and reveals a number of genes that may regulate MuSC/myoblast state transitions which had not been previously identified.

Tolerance to graded dosages of histidine supplementation in healthy human adults.

BACKGROUND: Histidine is an essential amino acid with health benefits that may warrant histidine supplementation; however, the clinical safety of histidine intake above the average dietary intake (1.52-5.20 g/d) needs to be vetted. OBJECTIVES: We aimed to determine the tolerance to graded dosages of histidine in a healthy adult population. METHODS: Healthy adults aged 21-50 y completed graded dosages of histidine supplement (4, 8, and 12 g/d, Study 1) (n = 20 men and n = 20 women) and/or a 16-g/d dosage of histidine (Study 2, n = 21 men and n = 19 women); 27 participants (n = 12 men and n = 15 women) completed both studies. After study enrollment and baseline measures, participants consumed encapsulated histidine for 4 wk followed by a 3-wk recovery period. Primary outcomes included vitals, select biochemical analytes, anthropometry, serum zinc, and body composition (via DXA). RESULTS: No changes in vitals or body composition occurred with histidine supplementation in either study. Plasma histidine (measured in subjects who completed all dosages for Studies 1 and 2) was elevated at the 12- and 16-g/d dosages (compared with 0-8 g/d, P < 0.05) and blood urea nitrogen increased with dosage (P = 0.013) and time (P < 0.001) in Study 1 and with time in Study 2 (P < 0.001). In Study 1, mean ferritin concentrations were lower in 12 g/d (46.0 ng/mL; 95% CI: 34.8, 60.9 ng/mL) than in 4 g/d (51.6 ng/mL; 95% CI: 39.0, 68.4 ng/mL; P = 0.038). In Study 2, 16 g/d increased mean aspartate aminotransferase from baseline (19 U/L; 95% CI: 17, 22 U/L) to week 4 (24 U/L; 95% CI: 21, 27 U/L; P < 0.001) and mean serum zinc decreased from baseline (0.75 µg/dL; 95% CI: 0.71, 0.80 µg/dL) to week 4 (0.70 µg/dL; 95% CI: 0.66, 0.74 µg/dL; P = 0.011). CONCLUSIONS: Although values remained within the normal reference ranges for all analytes measured, in all dosages tested, the human no-observed adverse effect level was determined to be 8 g/d owing to changes in blood parameters at the 12-g/d dosage.This trial was registered at clinicaltrials.gov as NCT04142294.

Consumption of a Blueberry-Enriched Diet by Women for 6 Weeks Alters Determinants of Human Muscle Progenitor Cell Function.

BACKGROUND: Human muscle progenitor cell (hMPC) function facilitates skeletal muscle regeneration and is influenced by circulating factors. Yet it is unknown whether dietary interventions impact hMPC function. Blueberry consumption was examined due to the pro-proliferative and antioxidant effects of blueberries and blueberry-derived compounds. OBJECTIVES: This study measured indicators of hMPC function in young and old cultures treated with serum collected from a blueberry-enriched diet (BED) intervention. METHODS: Younger (21-40 y, n = 12) and older (60-79 y, n = 10) women consumed a 6-wk BED (38 g of freeze-dried blueberries daily). Fasting serum was collected at 0, 4, and 6 wk, and a fed serum sample at 1.5 h (acute) after starting the BED intervention. Young and old hMPCs, derived from 3-5 distinct donors (biological replicates), were individually cultured in media containing pooled, age-group-matched serum from each time point. Determinants of hMPC function (e.g., hMPC number, oxidative stress resistance, and upregulation of metabolic pathways) were measured and compared within age groups. RESULTS: Culturing young hMPCs in acute (compared with 0 wk) BED serum did not alter hMPC number or oxidative stress-induced cell death, but increased cellular oxygen consumption (29%, P = 0.026). Culturing young hMPCs in 6-wk (compared with 0-wk) BED serum increased hMPC number (40%, P = 0.0024), conferred minor resistance to oxidative stress-induced cell death (12.6 percentage point decrease, P = 0.10), and modestly increased oxygen consumption (36%, P = 0.09). No beneficial effect of the acute or long-term BED serum was observed in old hMPCs. CONCLUSIONS: In younger women, dietary interventions could be a feasible strategy to improve hMPC function and thus muscle regeneration, through altering the serum environment.This study was registered at clinicaltrials.gov (NCT04262258).

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure.

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

Peptide YY (PYY) Is Expressed in Human Skeletal Muscle Tissue and Expanding Human Muscle Progenitor Cells.

Peptide YY (PYY) is considered a gut peptide with roles in post-prandial appetite and glucose regulation. Circulating PYY protein levels increase during aerobic exercise. Furthermore, people who have greater increases in muscle progenitor cells (hMPCs), the adult stem cell population responsible for skeletal muscle (SkM) repair, after resistance training have higher PYY transcript levels in SkM prior to training. Currently, examination of PYY expression patterns in SkM and/or hMPCs is lacking. Our objective was to identify the expression patterns of PYY in SkM and hMPCs. PYY and the associated Y receptors were analyzed in SkM biopsy tissue and cultured hMPCs from young and old human participants. Additional experiments to assess the role and regulation of PYY in hMPCs were performed. In SkM, PYY and one of the three Y receptors (Y1r) were detectable, but expression patterns were not affected by age. In expanding hMPCs, PYY and all three Y receptor (Y1r, Y2r, and Y5r) proteins were expressed in a temporal fashion with young hMPCs having greater levels of Y receptors at various time points. Exogenous PYY did not affect hMPC population expansion. hMPC PYY levels increased following the metabolic stimulus, 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), but were not affected by the inflammatory stimulus, tumor necrosis factor alpha (TNFα). In conclusion, PYY and Y receptor expression are not impacted by age in SkM tissue but are reduced in old vs. young expanding hMPCs. Furthermore, endogenous PYY production is stimulated by low energy states and thus may be integral for skeletal muscle and hMPC responses to metabolic stimuli.

Effect of dairy and nondairy snacks on postprandial blood glucose regulation in 9-14-year-old children.

In adults, dairy consumption improves short-term blood glucose regulation. It is unknown if these short-term benefits extend to children of different weight statuses. The objective of this study was to investigate the effect of a dairy and nondairy snack in both normal-weight (NW) and overweight/obese (OW/OB) children on blood glucose regulation and food intake (FI). In a repeated-measures crossover design, 11 NW and 7 OW/OB children (age: 9-14 years), consumed, in random order, a dairy (Greek yogurt, 198.9 g, 171 kcal, 0 g fat, 17 g protein) or nondairy (mini sandwich-type cookies, 37.5 g, 175 kcal, 7.5 g fat, 1.3 g protein) snack containing 25 g of available carbohydrates. Ad libitum FI was measured 120 min after snack consumption. Blood glucose, insulin, C-peptide, and glucagon-like peptide-1 (GLP-1) were measured at 0 min (before the snack), and at 30, 60, 90, and 120 min after snack consumption. Insulin secretion was calculated from deconvolution of C-peptide. Hepatic insulin extraction was calculated as C-peptide divided by insulin. FI did not differ between snacks (P = 0.55). Mean blood glucose was lower (P < 0.001) and insulin higher (P < 0.0001) in the 120 min after consuming the dairy snack. C-Peptide concentrations (P = 0.75) and insulin secretion (P = 0.37) were not different between snacks. The increase in insulin was explained by reduced hepatic insulin extraction (P < 0.01). Consumption of the dairy snack also increased mean GLP-1 concentrations (P < 0.001). In conclusion, consumption of a dairy snack by NW and OW/OB children results in reduced postprandial blood glucose concentrations and elevated circulating insulin compared with a nondairy snack possibly because of delayed hepatic insulin extraction.

Effect of video game playing and a glucose preload on subjective appetite, subjective emotions, and food intake in overweight and obese boys.

Video game playing (VGP) is associated with overweight/obesity (OW/OB). VGP and caloric preloads in the pre-meal environment influence short-term food intake (FI) in healthy-weight children. Therefore, the purpose of the present study was to examine the effect of pre-meal VGP and a glucose preload on subjective emotions, subjective appetite, and FI in boys with OW/OB. On 4 separate mornings, boys with OW/OB (n = 22; mean ± SD: age = 11.9 ± 1.6 years; body mass index percentile = 94.3 ± 3.9) participated in 4 test conditions. Two hours after a standardized breakfast, boys consumed equally sweetened preloads (250 mL) of sucralose (0 kcal) or glucose (200 kcal), with or without 30 min of subsequent VGP. Immediately after each test condition, FI was evaluated during an ad libitum pizza meal. Subjective appetite was measured at 0 (baseline), 15, and 30 min. Subjective emotions (aggression, anger, excitement, disappointment, happiness, upset, and frustration) were measured at 0 and 30 min. VGP did not affect FI, but the glucose preload decreased FI compared with the sucralose control (Δ = -103 ± 48 kcal, p < 0.01). However, cumulative FI (preload kcal + meal kcal) was 9% higher after the glucose preload (p < 0.01). Subjective appetite increased with time (p < 0.05) but was not influenced by preload or VGP. Frustration was the only subjective emotion that increased following VGP (p < 0.01). A glucose preload, but not VGP, suppressed FI in boys with OW/OB, suggesting a primary role of physiological factors in short-term FI regulation.

Understanding Age-Related Changes in Skeletal Muscle Metabolism: Differences Between Females and Males.

Skeletal muscle is the largest metabolic organ system in the human body. As such, metabolic dysfunction occurring in skeletal muscle impacts whole-body nutrient homeostasis. Macronutrient metabolism changes within the skeletal muscle with aging, and these changes are associated in part with age-related skeletal muscle remodeling. Moreover, age-related changes in skeletal muscle metabolism are affected differentially between males and females and are likely driven by changes in sex hormones. Intrinsic and extrinsic factors impact observed age-related changes and sex-related differences in skeletal muscle metabolism. Despite some support for sex-specific differences in skeletal muscle metabolism with aging, more research is necessary to identify underlying differences in mechanisms. Understanding sex-specific aging skeletal muscle will assist with the development of therapies to attenuate adverse metabolic and functional outcomes.