Lipidomics reveals diurnal lipid oscillations in human skeletal muscle persisting in cellular myotubes cultured in vitro.

Circadian clocks play an important role in lipid homeostasis, with impact on various metabolic diseases. Due to the central role of skeletal muscle in whole-body metabolism, we aimed at studying muscle lipid profiles in a temporal manner. Moreover, it has not been shown whether lipid oscillations in peripheral tissues are driven by diurnal cycles of rest-activity and food intake or are able to persist in vitro in a cell-autonomous manner. To address this, we investigated lipid profiles over 24 h in human skeletal muscle in vivo and in primary human myotubes cultured in vitro. Glycerolipids, glycerophospholipids, and sphingolipids exhibited diurnal oscillations, suggesting a widespread circadian impact on muscle lipid metabolism. Notably, peak levels of lipid accumulation were in phase coherence with core clock gene expression in vivo and in vitro. The percentage of oscillating lipid metabolites was comparable between muscle tissue and cultured myotubes, and temporal lipid profiles correlated with transcript profiles of genes implicated in their biosynthesis. Lipids enriched in the outer leaflet of the plasma membrane oscillated in a highly coordinated manner in vivo and in vitro. Lipid metabolite oscillations were strongly attenuated upon siRNA-mediated clock disruption in human primary myotubes. Taken together, our data suggest an essential role for endogenous cell-autonomous human skeletal muscle oscillators in regulating lipid metabolism independent of external synchronizers, such as physical activity or food intake.

Effect of resistance training and protein intake pattern on myofibrillar protein synthesis and proteome kinetics in older men in energy restriction.

KEY POINTS: Strategies to enhance the loss of fat while preserving muscle mass during energy restriction are of great importance to prevent sarcopenia in overweight older adults. We show for the first time that the integrated rate of synthesis of numerous individual contractile, cytosolic and mitochondrial skeletal muscle proteins was increased by resistance training (RT) and unaffected by dietary protein intake pattern during energy restriction in free-living, obese older men. We observed a correlation between the synthetic rates of skeletal muscle-derived proteins obtained in serum (creatine kinase M-type, carbonic anhydrase 3) and the synthetic rates of proteins obtained via muscle sampling; and that the synthesis rates of these proteins in serum revealed the stimulatory effects of RT. These results have ramifications for understanding the influence of RT on skeletal muscle and are consistent with the role of RT in maintaining muscle protein synthesis and potentially supporting muscle mass preservation during weight loss. ABSTRACT: We determined how the pattern of protein intake and resistance training (RT) influenced longer-term (2 weeks) integrated myofibrillar protein synthesis (MyoPS) during energy restriction (ER). MyoPS and proteome kinetics were measured during 2 weeks of ER alone and 2 weeks of ER plus RT (ER + RT) in overweight/obese older men. Participants were randomized to consume dietary protein in a balanced (BAL: 25% daily protein per meal × 4 meals) or skewed (SKEW: 7:17:72:4% daily protein per meal) pattern (n = 10 per group). Participants ingested deuterated water during the consecutive 2-week periods, and skeletal muscle biopsies and serum were obtained at the beginning and conclusion of ER and ER + RT. Bulk MyoPS (i.e. synthesis of the myofibrillar protein sub-fraction) and the synthetic rates of numerous individual skeletal muscle proteins were quantified. Bulk MyoPS was not affected by protein distribution during ER or ER + RT (ER: BAL = 1.24 ± 0.31%/day, SKEW = 1.26 ± 0.37%/day; ER + RT: BAL = 1.64 ± 0.48%/day, SKEW = 1.52 ± 0.66%/day) but was ∼26% higher during ER + RT than during ER (P = 0.023). The synthetic rates of 175 of 190 contractile, cytosolic and mitochondrial skeletal muscle proteins, as well as synthesis of muscle-derived proteins measured in serum, creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3), were higher during ER + RT than during ER (P < 0.05). In addition, the synthetic rates of CK-M and CA-3 measured in serum correlated with the synthetic rates of proteins obtained via muscle sampling (P < 0.05). This study provides novel data on the skeletal muscle adaptations to RT and dietary protein distribution.

Dietary protein intake in school-age children and detailed measures of body composition: the Generation R Study.

BACKGROUND: A high-protein diet in infancy increases the risk of obesity, but the effects of dietary protein intake in mid-childhood on body composition are unclear. Therefore, we studied associations of protein intake (total, animal and plant-sourced) at 8 years of age with anthropometric measures and body composition up to age 10 years. METHODS: We included 3991 children of the Generation R Study, a prospective cohort in the Netherlands. Dietary protein intake was assessed at 8 years of age using a food-frequency questionnaire and is expressed in energy percentage (E%). Anthropometric measures and body composition (using dual-energy X-ray absorptiometry (DXA)) were assessed at 6 years and during follow-up at 10 years. We calculated body mass index (BMI), fat mass index (FMI), and fat-free mass index (FFMI). All outcomes were sex- and age-standardized and overweight (yes/no) was derived from BMI-SDS. We examined associations of protein intake at 8 years with the combined risk of overweight and obesity, and body composition at 10 years using multivariable logistic and linear regression models. These analyses were adjusted for outcomes at 6 years and protein intake in early life. RESULTS: In multivariable-adjusted models, a 5E% higher protein intake at 8 years was associated with a higher combined risk of overweight and obesity up to 10 years (odds ratio (OR) 1.51, 95% confidence interval (CI): 1.22,1.86), independent of whether it replaced carbohydrates or fat. However, this was mainly explained by an association of protein intake with a higher FFMI (0.07 standard deviation scores (SDS) per 5E%, 95% CI: 0.02,0.11), not FMI. Both plant and animal were associated with a higher FFMI, but the association was stronger for protein from plant sources. For FMI, our findings also suggest trends of higher plant protein intake with lower FMI, and higher animal protein intake with higher FMI. Following this, a higher plant protein intake at the expense of animal protein was associated with a lower FMI (-0.08 SDS per 5E%, 95% CI: -0.15,-0.01). CONCLUSIONS: We observed that a higher protein intake in mid-childhood is associated with a higher fat-free mass. Our findings also suggest that protein from plant sources seems to be beneficial for body composition in school-age children.

Protein Intake at Breakfast Promotes a Positive Whole-Body Protein Balance in a Dose-Response Manner in Healthy Children: A Randomized Trial.

Background: Protein ingestion promotes whole-body net protein balance (NB) in children, which is a prerequisite for growth. Determining how much protein is required at breakfast to promote a positive NB, which may be negative after the traditional overnight fast in children, has yet to be determined. Objective: We determined the impact of incremental doses of milk protein at breakfast as well as the impact of daily dietary protein distribution on NB in children. Methods: A total of 28 children [14 boys, 14 girls; age range: 7-11 y; body mass index (mean ± SD, in kg/m2): 16.0 ± 1.9] completed 2 intervention trials. During the breakfast meal, participants consumed an isoenergetic beverage with different amounts of protein (0, 7, 14, or 21 g for Groups A-D, respectively) and [15N]-glycine to measure whole body protein metabolism. Whole-body nitrogen turnover, protein synthesis (PS), protein breakdown, and NB were measured over 9 and 24 h. Results: Following an overnight fast, children were in negative NB (-64.5 mg · kg-1 · h-1). Protein ingestion at breakfast induced a stepwise increase in NB over 9 h [Groups A (6.2 mg · kg-1 · h-1) < B (27.9 mg · kg-1 · h-1) < C (46.9 mg · kg-1 · h-1) < D (66.0 mg · kg-1 · h-1)] with all conditions different from each other (all P < 0.01). PS was 42% greater in Group D than in Group A over 9 h (P < 0.05). Conclusions: Consuming ≥7 g of the total daily protein intake at breakfast attenuates the observed overnight protein losses in children during the subsequent 9 h following breakfast consumption. The dose-dependent increase in NB over a daytime fed period, inclusive of breakfast and lunch, highlights the importance of breakfast protein intake on acute anabolism in healthy active children. This trial was registered at clinicaltrials.gov as NCT02465151.

Protein and Energy Intakes Are Skewed toward the Evening among Children and Adolescents in the United States: NHANES 2013-2014.

Background: Emerging evidence suggests that the timing, amount at individual eating occasions, and distribution of protein and energy intakes throughout the day may affect health.Objective: We examined the timing, amounts, and distribution of protein and energy intakes throughout the day among participants aged 4-18 y in the United States in the context of chronobiology and nutrition.Methods: This cross-sectional analysis included 2532 participants aged 4-18 y who completed the first interviewer-administered 24-h dietary recall in NHANES 2013-2014. Descriptive statistics for intakes across the day were provided as percentiles, means ± SEMs, and percentages of nonconsumers. Statistical differences between intakes across the day were tested with the use of individual-level fixed-effects regression models. Cumulative distribution functions were used to examine the timing of the first and last caloric eating occasion.Results: Mean ± SEM protein (grams) and energy (percentage of the day) intakes were significantly higher (P < 0.05) in the evening than in the morning among all age groups. The percentage of participants aged 4-8, 9-13, and 14-18 y who had their first eating occasion at or after 1100 was 4%, 14%, and 20%, respectively, and the percentage who had their last eating occasion at or after 2100 was 8%, 19%, and 34%, respectively.Conclusions: Protein and energy intakes among participants aged 4-18 y in this study were largest in the evening and midday and smallest in the morning and afternoon. Clinical trials are needed to assess any potential impact such dietary behaviors may have on health outcomes related to metabolic dysfunction in children and adolescents.

A Systematic Review of the Effects of Plant Compared with Animal Protein Sources on Features of Metabolic Syndrome.

Dietary protein may play an important role in the prevention of metabolic dysfunctions. However, the way in which the protein source affects these dysfunctions has not been clearly established. The aim of the current systematic review was to compare the impact of plant- and animal-sourced dietary proteins on several features of metabolic syndrome in humans. The PubMed database was searched for both chronic and acute interventional studies, as well as observational studies, in healthy humans or those with metabolic dysfunctions, in which the impact of animal and plant protein intake was compared while using the following variables: cholesterolemia and triglyceridemia, blood pressure, glucose homeostasis, and body composition. Based on data extraction, we observed that soy protein consumption (with isoflavones), but not soy protein alone (without isoflavones) or other plant proteins (pea and lupine proteins, wheat gluten), leads to a 3% greater decrease in both total and LDL cholesterol compared with animal-sourced protein ingestion, especially in individuals with high fasting cholesterol concentrations. This observation was made when animal proteins were provided as a whole diet rather than given supplementally. Some observational studies reported an inverse association between plant protein intake and systolic and diastolic blood pressure, but this was not confirmed by intervention studies. Moreover, plant protein (wheat gluten, soy protein) intake as part of a mixed meal resulted in a lower postprandial insulin response than did whey. This systematic review provides some evidence that the intake of soy protein associated with isoflavones may prevent the onset of risk factors associated with cardiovascular disease, i.e., hypercholesterolemia and hypertension, in humans. However, we were not able to draw any further conclusions from the present work on the positive effects of plant proteins relating to glucose homeostasis and body composition.

Postexercise Dietary Protein Ingestion Increases Whole-Body Leucine Balance in a Dose-Dependent Manner in Healthy Children.

Background: Protein ingestion is important in enhancing whole-body protein balance in children. The effect of discrete bolus protein ingestion on acute postexercise recovery has yet to be determined.Objective: This study determined the effect of increasing doses of ingested protein on postexercise whole-body leucine balance in healthy, active children.Methods: Thirty-five children (26 boys, 9 girls; age range: 9-13 y; weight mean ± SD: 44.9 ± 10.6 kg) underwent a 5-d adaptation diet (0.95 g protein ⋅ kg-1 ⋅ d-1) before performing 20 min of cycling 3 times with a concurrent, primed, constant infusion of [13C]leucine. After exercise, participants consumed an isoenergetic beverage (140 kcal) containing variable amounts of bovine skim-milk protein and carbohydrates (sucrose) (0, 5, 10, and 15 g protein made up with 35, 30, 25, and 20 g carbohydrates, respectively). Blood and breath samples were taken over the 3 h of recovery to determine non-steady state whole-body leucine oxidation (LeuOX) and net leucine balance (LeuBAL).Results: LeuOX (secondary outcome) peaked 60 min after beverage ingestion and demonstrated a relative dose-response over the 3 h of recovery (15 g = 10 > 5 > 0 g; P < 0.001). LeuBAL (primary outcome) demonstrated a dose-response over the 3 h [15 g (24.2 ± 8.2 mg/kg) > 10 g (11.6 ± 4.3 mg/kg) > 5 g (5.7 ± 1.9 mg/kg) > 0 g (-3.0 ± 1.7 mg/kg); all P < 0.01] with all conditions different from zero (all P < 0.001).Conclusions: Over the 3-h postexercise period, LeuBAL was negative with carbohydrate ingestion alone; however, the co-ingestion of carbohydrates and 5 g high-quality dietary protein was sufficient to promote a positive postexercise whole-body protein balance in healthy, active children. Moreover, LeuBAL increased in a dose-dependent manner within the protein range studied. Children should consider consuming a source of dietary protein after physical activity to enhance whole-body anabolism. This trial was registered at clinicaltrials.gov as NCT01598935.

Protein ingestion preserves proteasome activity during intense aseptic inflammation and facilitates skeletal muscle recovery in humans.

The ubiquitin-proteasome system (UPS) is the main cellular proteolytic system responsible for the degradation of normal and abnormal (e.g. oxidised) proteins. Under catabolic conditions characterised by chronic inflammation, the UPS is activated resulting in proteolysis, muscle wasting and impaired muscle function. Milk proteins provide sulphur-containing amino acid and have been proposed to affect muscle inflammation. However, the response of the UPS to aseptic inflammation and protein supplementation is largely unknown. The aim of this study was to investigate how milk protein supplementation affects UPS activity and skeletal muscle function under conditions of aseptic injury induced by intense, eccentric exercise. In a double-blind, cross-over, repeated measures design, eleven men received either placebo (PLA) or milk protein concentrate (PRO, 4×20 g on exercise day and 20 g/d for the following 8 days), following an acute bout of eccentric exercise (twenty sets of fifteen eccentric contractions at 30°/s) on an isokinetic dynamometer. In each trial, muscle biopsies were obtained from the vastus lateralis muscle at baseline, as well as at 2 and 8 d post exercise, whereas blood samples were collected before exercise and at 6 h, 1 d, 2 d and 8 d post exercise. Muscle strength and soreness were assessed before exercise, 6 h post exercise and then daily for 8 consecutive days. PRO preserved chymotrypsin-like activity and attenuated the decrease of strength, facilitating its recovery. PRO also prevented the increase of NF-κB phosphorylation and HSP70 expression throughout recovery. We conclude that milk PRO supplementation following exercise-induced muscle trauma preserves proteasome activity and attenuates strength decline during the pro-inflammatory phase.

Inflammaging and Skeletal Muscle: Can Protein Intake Make a Difference?

Inflammaging is the chronic low-grade inflammatory state present in the elderly, characterized by increased systemic concentrations of proinflammatory cytokines. It has been shown that inflammaging increases the risk of pathologic conditions and age-related diseases, and that it also has been associated with increased skeletal muscle wasting, strength loss, and functional impairments. Experimental evidence suggests that the increased concentrations of proinflammatory cytokines and primary tumor necrosis factor α observed in chronic inflammation lead to protein degradation through proteasome activation and reduced skeletal muscle protein synthesis (MPS) via protein kinase B/Akt downregulation. Dairy and soy proteins contain all the essential amino acids, demonstrate sufficient absorption kinetics, and include other bioactive peptides that may offer nutritional benefits, in addition to those of stimulating MPS. Whey protein has antioxidative effects, primarily because of its ability to enhance the availability of reduced glutathione and the activity of the endogenous antioxidative enzyme system. Soy protein and isoflavone-enriched soy protein, meanwhile, may counteract chronic inflammation through regulation of the nuclear transcription factor κB signaling pathway and cytokine production. Although evidence suggests that whey protein, soy protein, and isoflavone-enriched soy proteins may be promising nutritional interventions against the oxidative stress and chronic inflammation present in pathologic conditions and aging (inflammaging), there is a lack of information about the anabolic potential of dietary protein intake and protein supplementation in elderly people with increased systemic inflammation. The antioxidative and anti-inflammatory effects, as well as the anabolic potential of protein supplementation, should be further investigated in the future with well-designed clinical trials focusing on inflammaging and its associated skeletal muscle loss.

Screen-based sedentary behavior and associations with functional strength in 6-15 year-old children in the United States.

BACKGROUND: Physical strength is associated with improved health outcomes in children. Heavier children tend to have lower functional strength and mobility. Physical activity can increase children's strength, but it is unknown how different types of electronic media use impact physical strength. METHODS: Data from the NHANES National Youth Fitness Survey (NNYFS) from children ages 6-15 were analyzed in this study. Regression models were conducted to determine if screen-based sedentary behaviors (television viewing time, computer/video game time) were associated with strength measures (grip, leg extensions, modified pull-ups, plank) while controlling for potential confounders including child age, sex, BMI z-score, and days per week with 60+ minutes of physical activity. Grip strength and leg extensions divided by body weight were analyzed to provide measures of relative strength together with pull-ups and plank, which require lifting the body. RESULTS: The results from the regression models showed the hypothesized inverse association between TV time and all strength measures. Computer time was only significantly inversely associated with the ability to do one or more pull-ups. CONCLUSIONS: This study shows that television viewing, but not computer/videogames, is inversely associated with measures of child strength while controlling for child characteristics and physical activity. These findings suggest that "screen time" may not be a unified construct with respect to strength outcomes and that further exploration of the potential benefits of reducing television time on children's strength and related mobility is needed.

Hypoenergetic diet-induced reductions in myofibrillar protein synthesis are restored with resistance training and balanced daily protein ingestion in older men.

Strategies to enhance weight loss with a high fat-to-lean ratio in overweight/obese older adults are important since lean loss could exacerbate sarcopenia. We examined how dietary protein distribution affected muscle protein synthesis during energy balance (EB), energy restriction (ER), and energy restriction plus resistance training (ER + RT). A 4-wk ER diet was provided to overweight/obese older men (66 ± 4 yr, 31 ± 5 kg/m(2)) who were randomized to either a balanced (BAL: 25% daily protein/meal × 4) or skewed (SKEW: 7:17:72:4% daily protein/meal; n = 10/group) pattern. Myofibrillar and sarcoplasmic protein fractional synthetic rates (FSR) were measured during a 13-h primed continuous infusion of l-[ring-(13)C6]phenylalanine with BAL and SKEW pattern of protein intake in EB, after 2 wk ER, and after 2 wk ER + RT. Fed-state myofibrillar FSR was lower in ER than EB in both groups (P < 0.001), but was greater in BAL than SKEW (P = 0.014). In ER + RT, fed-state myofibrillar FSR increased above ER in both groups and in BAL was not different from EB (P = 0.903). In SKEW myofibrillar FSR remained lower than EB (P = 0.002) and lower than BAL (P = 0.006). Fed-state sarcoplasmic protein FSR was reduced similarly in ER and ER + RT compared with EB (P < 0.01) in both groups. During ER in overweight/obese older men a BAL consumption of protein stimulated the synthesis of muscle contractile proteins more effectively than traditional, SKEW distribution. Combining RT with a BAL protein distribution "rescued" the lower rates of myofibrillar protein synthesis during moderate ER.

Mitochondria as Nutritional Targets to Maintain Muscle Health and Physical Function During Ageing.

The age-related loss of skeletal muscle mass and physical function leads to a loss of independence and an increased reliance on health-care. Mitochondria are crucial in the aetiology of sarcopenia and have been identified as key targets for interventions that can attenuate declines in physical capacity. Exercise training is a primary intervention that reduces many of the deleterious effects of ageing in skeletal muscle quality and function. However, habitual levels of physical activity decline with age, making it necessary to implement adjunct treatments to maintain skeletal muscle mitochondrial health and physical function. This review provides an overview of the effects of ageing and exercise training on human skeletal muscle mitochondria and considers several supplements that have plausible mechanistic underpinning to improve physical function in ageing through their interactions with mitochondria. Several supplements, including MitoQ, urolithin A, omega-3 polyunsaturated fatty acids (n3-PUFAs), and a combination of glycine and N-acetylcysteine (GlyNAC) can improve physical function in older individuals through a variety of inter-dependent mechanisms including increases in mitochondrial biogenesis and energetics, decreases in mitochondrial reactive oxygen species emission and oxidative damage, and improvements in mitochondrial quality control. While there is evidence that some nicotinamide adenine dinucleotide precursors can improve physical function in older individuals, such an outcome seems unrelated to and independent of changes in skeletal muscle mitochondrial function. Future research should investigate the safety and efficacy of compounds that can improve skeletal muscle health in preclinical models through mechanisms involving mitochondria, such as mitochondrial-derived peptides and mitochondrial uncouplers, with a view to extending the human health-span.

Skeletal muscle myostatin mRNA expression is upregulated in aged human adults with excess adiposity but is not associated with insulin resistance and ageing.

Myostatin negatively regulates skeletal muscle growth and appears upregulated in human obesity and associated with insulin resistance. However, observations are confounded by ageing, and the mechanisms responsible are unknown. The aim of this study was to delineate between the effects of excess adiposity, insulin resistance and ageing on myostatin mRNA expression in human skeletal muscle and to investigate causative factors using in vitro models. An in vivo cross-sectional analysis of human skeletal muscle was undertaken to isolate effects of excess adiposity and ageing per se on myostatin expression. In vitro studies employed human primary myotubes to investigate the potential involvement of cross-talk between subcutaneous adipose tissue (SAT) and skeletal muscle, and lipid-induced insulin resistance. Skeletal muscle myostatin mRNA expression was greater in aged adults with excess adiposity than age-matched adults with normal adiposity (2.0-fold higher; P < 0.05) and occurred concurrently with altered expression of genes involved in the maintenance of muscle mass but did not differ between younger and aged adults with normal adiposity. Neither chronic exposure to obese SAT secretome nor acute elevation of fatty acid availability (which induced insulin resistance) replicated the obesity-mediated upregulation of myostatin mRNA expression in vitro. In conclusion, skeletal muscle myostatin mRNA expression is uniquely upregulated in aged adults with excess adiposity and insulin resistance but not by ageing alone. This does not appear to be mediated by the SAT secretome or by lipid-induced insulin resistance. Thus, factors intrinsic to skeletal muscle may be responsible for the obesity-mediated upregulation of myostatin, and future work to establish causality is required.

Characterising 24-h skeletal muscle gene expression alongside metabolic & endocrine responses under diurnal conditions.

CONTEXT: Skeletal muscle plays a central role in the storage, synthesis, and breakdown of nutrients, yet little research has explored temporal responses of this human tissue, especially with concurrent measures of systemic biomarkers of metabolism. OBJECTIVE: To characterise temporal profiles in skeletal muscle expression of genes involved in carbohydrate metabolism, lipid metabolism, circadian clocks, and autophagy and descriptively relate them to systemic metabolites and hormones during a controlled laboratory protocol. METHODS: Ten healthy adults (9M/1F, mean ± SD: age: 30 ± 10 y; BMI: 24.1 ± 2.7 kg·m-2) rested in the laboratory for 37 hours with all data collected during the final 24 hours of this period (i.e., 0800-0800 h). Participants ingested hourly isocaloric liquid meal replacements alongside appetite assessments during waking before a sleep opportunity from 2200-0700 h. Blood samples were collected hourly for endocrine and metabolite analyses, with muscle biopsies occurring every 4 h from 1200 h to 0800 h the following day to quantify gene expression. RESULTS: Plasma insulin displayed diurnal rhythmicity peaking at 1804 h. Expression of skeletal muscle genes involved in carbohydrate metabolism (Name - Acrophase; GLUT4 - 1440 h; PPARGC1A -1613 h; HK2 - 1824 h) and lipid metabolism (FABP3 - 1237 h; PDK4 - 0530 h; CPT1B - 1258 h) displayed 24 h rhythmicity that reflected the temporal rhythm of insulin. Equally, circulating glucose (0019 h), NEFA (0456 h), glycerol (0432 h), triglyceride (2314 h), urea (0046 h), CTX (0507 h) and cortisol concentrations (2250 h) also all displayed diurnal rhythmicity. CONCLUSION: Diurnal rhythms were present in human skeletal muscle gene expression as well systemic metabolites and hormones under controlled diurnal conditions. The temporal patterns of genes relating to carbohydrate and lipid metabolism alongside circulating insulin are consistent with diurnal rhythms being driven in part by the diurnal influence of cyclic feeding and fasting.

Trigonelline is an NAD+ precursor that improves muscle function during ageing and is reduced in human sarcopenia.

Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD+) levels are hallmarks of skeletal muscle ageing and sarcopenia1-3, but it is unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we report a functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid4, NAD+ levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally occurring and isotopically labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD+ pool and increases NAD+ levels in Caenorhabditis elegans, mice and primary myotubes from healthy individuals and individuals with sarcopenia. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the nicotinate phosphoribosyltransferase/Preiss-Handler pathway5,6 across models. In C. elegans, trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD+-dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during ageing. Collectively, we identify nutritional supplementation of trigonelline as an NAD+-boosting strategy with therapeutic potential for age-associated muscle decline.

A meta-analysis comparing the effectiveness of alternate day fasting, the 5:2 diet, and time-restricted eating for weight loss.

OBJECTIVE: The objective of this meta-analysis was to compare the effectiveness of different intermittent fasting (IF) regimens on weight loss, in the general population, and compare these to traditional caloric energy restriction (CER). METHODS: Three databases were searched from 2011 to June 2021 for randomized controlled trials (RCTs) that assessed weight loss and IF, including alternate day fasting (ADF), the 5:2 diet, and time-restricted eating (TRE). A random effect network analysis was used to compare the effectiveness between the three regimens. Meta-regression analysis was presented as weighted mean differences of body weight loss. RESULTS: The exploratory random effects network analysis of 24 RCTs (n = 1768) ranked ADF as the most effective, followed by CER and TRE. The meta-analysis showed that IF regimens resulted in similar weight loss to CER (mean difference 0.26 kg, 95% CI: -0.31 to 0.84; p = 0.37). Compliance was generally high (>80%) in trials shorter than 3 months. CONCLUSIONS: The present meta-analysis concludes that IF is comparable to CER and a promising alternative for weight loss. Among the three regimens, ADF showed the highest effectiveness for weight loss, followed by CER and TRE. Further well-powered RCTs with longer durations of intervention are required to draw solid conclusions.

Impact of moderate dietary protein restriction on glucose homeostasis in a model of estrogen deficiency.

The need to consume adequate dietary protein to preserve physical function during ageing is well recognized. However, the effect of protein intakes on glucose metabolism is still intensively debated. During age-related estrogen withdrawal at the time of the menopause, it is known that glucose homeostasis may be impaired but the influence of dietary protein levels in this context is unknown. The aim of the present study is to elucidate the individual and interactive effects of estrogen deficiency and suboptimal protein intake on glucose homeostasis in a preclinical model involving ovariectomy (OVX) and a 13 week period of a moderately reduced protein intake in 7 month-old ageing rats. To investigate mechanisms of action acting via the pancreas-liver-muscle axis, fasting circulating levels of insulin, glucagon, IGF-1, FGF21 and glycemia were measured. The hepatic lipid infiltration and the protein expression of GLUT4 in the gastrocnemius were analyzed. The gene expression of some hepatokines, myokines and lipid storage/oxidation related transcription factors were quantified in the liver and the gastrocnemius. We show that, regardless of the estrogen status, moderate dietary protein restriction increases fasting glycemia without modifying insulinemia, body weight gain and composition. This fasting hyperglycemia is associated with estrogen status-specific metabolic alterations in the muscle and liver. In estrogen-replete (SHAM) rats, GLUT4 was down-regulated in skeletal muscle while in estrogen-deficient (OVX) rats, hepatic stress-associated hyperglucagonemia and high serum FGF21 were observed. These findings highlight the importance of meeting dietary protein needs to avoid disturbances in glucose homeostasis in ageing female rats with or without estrogen withdrawal.

A Randomized Controlled Clinical Trial in Healthy Older Adults to Determine Efficacy of Glycine and N-Acetylcysteine Supplementation on Glutathione Redox Status and Oxidative Damage.

Glycine and cysteine are non-essential amino acids that are required to generate glutathione, an intracellular tripeptide that neutralizes reactive oxygen species and prevents tissue damage. During aging glutathione demand is thought to increase, but whether additional dietary intake of glycine and cysteine contributes towards the generation of glutathione in healthy older adults is not well understood. We investigated supplementation with glycine and n-acetylcysteine (GlyNAC) at three different daily doses for 2 weeks (low dose: 2.4 g, medium dose: 4.8 g, or high dose: 7.2 g/day, 1:1 ratio) in a randomized, controlled clinical trial in 114 healthy volunteers. Despite representing a cohort of healthy older adults (age mean = 65 years), we found significantly higher baseline levels of markers of oxidative stress, including that of malondialdehyde (MDA, 0.158 vs. 0.136 µmol/L, p < 0.0001), total cysteine (Cysteine-T, 314.8 vs. 276 µM, p < 0.0001), oxidized glutathione (GSSG, 174.5 vs. 132.3 µmol/L, p < 0.0001), and a lower ratio of reduced to oxidized glutathione (GSH-F:GSSG) (11.78 vs. 15.26, p = 0.0018) compared to a young reference group (age mean = 31.7 years, n = 20). GlyNAC supplementation was safe and well tolerated by the subjects, but did not increase levels of GSH-F:GSSG (end of study, placebo = 12.49 vs. 7.2 g = 12.65, p-value = 0.739) or that of total glutathione (GSH-T) (end of study, placebo = 903.5 vs. 7.2 g = 959.6 mg/L, p-value = 0.278), the primary endpoint of the study. Post-hoc analyses revealed that a subset of subjects characterized by high oxidative stress (above the median for MDA) and low baseline GSH-T status (below the median), who received the medium and high doses of GlyNAC, presented increased glutathione generation (end of study, placebo = 819.7 vs. 4.8g/7.2 g = 905.4 mg/L, p-value = 0.016). In summary GlyNAC supplementation is safe, well tolerated, and may increase glutathione levels in older adults with high glutathione demand. Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT05041179, NCT05041179.

Unacylated ghrelin, leptin, and appetite display diurnal rhythmicity in lean adults.

Constant routine and forced desynchrony protocols typically remove the effects of behavioral/environmental cues to examine endogenous circadian rhythms, yet this may not reflect rhythms of appetite regulation in the real world. It is therefore important to understand these rhythms within the same subjects under controlled diurnal conditions of light, sleep, and feeding. Ten healthy adults (9 M/1 F, means ±SD: age, 30 ± 10 yr; body mass index, 24.1 ± 2.7 kg·m-2) rested supine in the laboratory for 37 h. All data were collected during the final 24 h of this period (i.e., 0800-0800 h). Participants were fed hourly isocaloric liquid meal replacements alongside appetite assessments during waking before a sleep opportunity from 2200 to 0700 h. Hourly blood samples were collected throughout the 24-h period. Dim light melatonin onset occurred at 2318 ± 46 min. A diurnal rhythm in mean plasma unacylated ghrelin concentration was identified (P = 0.04), with the acrophase occurring shortly after waking (0819), falling to a nadir in the evening with a relative amplitude of 9%. Plasma leptin concentration also exhibited a diurnal rhythm (P < 0.01), with the acrophase occurring shortly after lights-out (0032 h) and the lowest concentrations at midday. The amplitude for this rhythm was 25%. Diurnal rhythms were established in all dimensions of appetite except for sweet preference (P = 0.29), with both hunger (2103 h) and prospective food consumption (1955 h) reaching their peak in the evening before falling to their nadir shortly after waking. Under controlled diurnal conditions, simultaneous measurement of leptin, unacylated ghrelin, and subjective appetite over a 24-h period revealed rhythmicity in appetite regulation in lean, healthy humans.NEW & NOTEWORTHY Simultaneous assessment of subjective appetite, unacylated ghrelin, and leptin was carried out over a continuous 37-h protocol for the first time under conditions of controlled light, sleep, and feeding in healthy, lean adults. Rhythms were observed in unacylated ghrelin, leptin, and components of subjective appetite, such as hunger, prospective consumption, and fullness. Concurrent measurement of rhythms in these variables is important to fully understand the temporal relationships between components of appetite as well as the influence of diurnal factors such as sleep, light, and feeding.