Liver-derived plasminogen mediates muscle stem cell expansion during caloric restriction through the plasminogen receptor Plg-RKT.

An intriguing effect of short-term caloric restriction (CR) is the expansion of certain stem cell populations, including muscle stem cells (satellite cells), which facilitate an accelerated regenerative program after injury. Here, we utilized the MetRSL274G (MetRS) transgenic mouse to identify liver-secreted plasminogen as a candidate for regulating satellite cell expansion during short-term CR. Knockdown of circulating plasminogen prevents satellite cell expansion during short-term CR. Furthermore, loss of the plasminogen receptor KT (Plg-RKT) is also sufficient to prevent CR-related satellite cell expansion, consistent with direct signaling of plasminogen through the plasminogen receptor Plg-RKT/ERK kinase to promote proliferation of satellite cells. Importantly, we are able to replicate many of these findings in human participants from the CALERIE trial. Our results demonstrate that CR enhances liver protein secretion of plasminogen, which signals directly to the muscle satellite cell through Plg-RKT to promote proliferation and subsequent muscle resilience during CR.

Causal analysis identifies small HDL particles and physical activity as key determinants of longevity of older adults.

BACKGROUND: The hard endpoint of death is one of the most significant outcomes in both clinical practice and research settings. Our goal was to discover direct causes of longevity from medically accessible data. METHODS: Using a framework that combines local causal discovery algorithms with discovery of maximally predictive and compact feature sets (the "Markov boundaries" of the response) and equivalence classes, we examined 186 variables and their relationships with survival over 27 years in 1507 participants, aged ≥71 years, of the longitudinal, community-based D-EPESE study. FINDINGS: As few as 8-15 variables predicted longevity at 2-, 5- and 10-years with predictive performance (area under receiver operator characteristic curve) of 0·76 (95% CIs 0·69, 0·83), 0·76 (0·72, 0·81) and 0·66 (0·61, 0·71), respectively. Numbers of small high-density lipoprotein particles, younger age, and fewer pack years of cigarette smoking were the strongest determinants of longevity at 2-, 5- and 10-years, respectively. Physical function was a prominent predictor of longevity at all time horizons. Age and cognitive function contributed to predictions at 5 and 10 years. Age was not among the local 2-year prediction variables (although significant in univariable analysis), thus establishing that age is not a direct cause of 2-year longevity in the context of measured factors in our data that determine longevity. INTERPRETATION: The discoveries in this study proceed from causal data science analyses of deep clinical and molecular phenotyping data in a community-based cohort of older adults with known lifespan. FUNDING: NIH/NIA R01AG054840, R01AG12765, and P30-AG028716, NIH/NIA Contract N01-AG-12102 and NCRR 1UL1TR002494-01.

Differential microRNA profiles of intramuscular and secreted extracellular vesicles in human tissue-engineered muscle.

Exercise affects the expression of microRNAs (miR/s) and muscle-derived extracellular vesicles (EVs). To evaluate sarcoplasmic and secreted miR expression in human skeletal muscle in response to exercise-mimetic contractile activity, we utilized a three-dimensional tissue-engineered model of human skeletal muscle ("myobundles"). Myobundles were subjected to three culture conditions: no electrical stimulation (CTL), chronic low frequency stimulation (CLFS), or intermittent high frequency stimulation (IHFS) for 7 days. RNA was isolated from myobundles and from extracellular vesicles (EVs) secreted by myobundles into culture media; miR abundance was analyzed by miRNA-sequencing. We used edgeR and a within-sample design to evaluate differential miR expression and Pearson correlation to evaluate correlations between myobundle and EV populations within treatments with statistical significance set at p < 0.05. Numerous miRs were differentially expressed between myobundles and EVs; 116 miRs were differentially expressed within CTL, 3 within CLFS, and 2 within IHFS. Additionally, 25 miRs were significantly correlated (18 in CTL, 5 in CLFS, 2 in IHFS) between myobundles and EVs. Electrical stimulation resulted in differential expression of 8 miRs in myobundles and only 1 miR in EVs. Several KEGG pathways, known to play a role in regulation of skeletal muscle, were enriched, with differentially overrepresented miRs between myobundle and EV populations identified using miEAA. Together, these results demonstrate that in vitro exercise-mimetic contractile activity of human engineered muscle affects both their expression of miRs and number of secreted EVs. These results also identify novel miRs of interest for future studies of the role of exercise in organ-organ interactions in vivo.

Epigenome-wide Association Study Analysis of Calorie Restriction in Humans, CALERIETM Trial Analysis.

Calorie restriction (CR) increases healthy life span and is accompanied by slowing or reversal of aging-associated DNA methylation (DNAm) changes in animal models. In the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIETM) human trial, we evaluated associations of CR and changes in whole-blood DNAm. CALERIETM randomized 220 healthy, nonobese adults in a 2:1 allocation to 2 years of CR or ad libitum (AL) diet. The average CR in the treatment group through 24 months of follow-up was 12%. Whole blood (baseline, 12, and 24 months) DNAm profiles were measured. Epigenome-wide association study (EWAS) analysis tested CR-induced changes from baseline to 12 and 24 months in the n = 197 participants with available DNAm data. CR treatment was not associated with epigenome-wide significant (false discovery rate [FDR] < 0.05) DNAm changes at the individual-CpG-site level. Secondary analysis of sets of CpG sites identified in published EWAS revealed that CR induced DNAm changes opposite to those associated with higher body mass index and cigarette smoking (p < .003 at 12- and 24-month follow-ups). In contrast, CR altered DNAm at chronological-age-associated CpG sites in the direction of older age (p < .003 at 12- and 24-month follow-ups). Although individual CpG site DNAm changes in response to CR were not identified, analyses of sets CpGs identified in prior EWAS revealed CR-induced changes to blood DNAm. Altered CpG sets were enriched for insulin production, glucose tolerance, inflammation, and DNA-binding and DNA-regulation pathways, several of which are known to be modified by CR. DNAm changes may contribute to CR effects on aging.

Differential effects of calorie restriction and rapamycin on age-related molecular and functional changes in skeletal muscle.

Aging is a multifactorial process associated with progressive degradation of physiological integrity and function. One of the greatest factors contributing to the deleterious effects of aging is the decline of functional ability due to loss of muscle mass, strength, and function, a condition termed sarcopenia. Calorie restriction (CR) has consistently been shown to extend lifespan and delay the onset and progression of various age-related diseases, including sarcopenia. Additional anti-aging interventions that are receiving scientific attention are CR mimetics. Of these pharmacological compounds, rapamycin has shown similar CR-related longevity benefits without the need for diet restrictions. To investigate the potential role of rapamycin as an anti-sarcopenic alternative to CR, we conducted a study in male and female C57BL/6 J mice to assess the effects of rapamycin on age-related gene expression changes in skeletal muscle associated with loss of muscle mass, strength, and function, relative to control. We hypothesize that the effects of rapamycin will closely align with CR with respect to physical function and molecular indices associated with muscle quality. Our results indicate CR and rapamycin provide partial protection against age-related decline in muscle, while engaging uniquely different molecular pathways in skeletal muscle. Our preclinical findings of the therapeutic potential of rapamycin or a CR regimen on geroprotective benefits in muscle should be extended to translational studies towards the development of effective strategies for the prevention and management of sarcopenia.

Psychometrics of the Balance Beam Functional Test in C57BL/6 Mice.

Aging is associated with a progressive decline in physical function characterized by decreased mobility, which is an important risk factor for loss of independence and reduced quality of life. Functional testing conducted in animals has advanced our understanding of age-related changes in physical ability and contributed to the development of physiologic measurements that can be used to assess functional changes during aging. The balance beam test is one assessment tool used to measure age-related changes in balance and coordination. The goal of this study is to provide analytical examples and psychometric support of a protocol that has been analyzed to show how the number of successive test runs, foot slips, pauses, and hesitations affect the reliability of the primary outcome measure, which is the time to cross the beam. Our results suggest that conducting more than 1 training session, consisting of greater than or equal to 3 successful training runs, followed by at least one test session with no less than 2 successful runs (that is, runs without pauses or hesitations) provides a psychometrically sound outcome. The data presented here indicate that a psychometric approach can improve protocol design and reliability of balance beam measures in mice.

Rationale and Design for a Higher (Dairy) Protein Weight Loss Intervention That Promotes Muscle Quality and Bone Health in Older Adults with Obesity: A Randomized, Controlled Pilot Study.

In contrast to recommendations for young and middle-aged adults, intentional weight loss among older adults remains controversial and is inconsistently advised. Recent research suggests that a higher protein diet can mitigate loss of lean mass during periods of intentional weight loss among older adults with obesity; however, the effects of intentional weight loss on skeletal muscle and bone are not fully understood. The Dairy in the Diet Yields New Approaches for Muscle Optimization (DDYNAMO) trial is a 6-month, randomized, controlled pilot study assessing the effects of combining regular, generous intakes of high quality protein (30 g/meal; primarily from dairy) with caloric restriction (-500kcal/d) and low-intensity resistance exercise (30 min/3 times per week) on muscle quality, muscle composition, bone mineral density in men and women aged ≥60 years with obesity and mild to moderate functional impairment (Short Physical Performance Battery [SPPB] score ≥4 to ≤10). Participants will be re-assessed at 18 months to evaluate weight maintenance, bone mineral density, physical function, and other secondary measures. ClinicalTrials.gov Identifier: NCT02437643.

Impact on cardiometabolic risk of a weight loss intervention with higher protein from lean red meat: Combined results of 2 randomized controlled trials in obese middle-aged and older adults.

BACKGROUND: The recognized benefits of a higher protein diet on muscle mass and strength in older adults are tempered by concerns of the potentially negative cardiometabolic impact of dietary sources of animal protein. OBJECTIVE: The aim of this study was to explore the cardiometabolic impact of 2 weight reduction diets: a higher protein diet, providing balanced portions of lean beef and pork throughout the day, vs. a diet following the Recommended Daily Allowance level of protein in obese middle-aged and older adults. METHODS: Data from Measuring Eating, Activity and Strength: Understanding the Response-Using Protein and Protein Optimization in Women Enables Results-Using Protein were combined for the present analysis. Subjects were randomly assigned to a 6-month weight loss diet (500 kcal deficit) and prescribed a Recommended Daily Allowance level of protein (0.8 g protein/kg BW), control group, or a higher level of protein (1.2 g protein/kg BW), protein group. For the protein group, lean, high-quality protein was evenly distributed between meals or balanced throughout the day (30 g protein/meal). The following cardiometabolic markers were quantified by nuclear magnetic resonance spectroscopy: lipids, lipoproteins, GlycA, trimethylamine-N-oxide, betaine, branched-chain amino acids, and lipoprotein insulin resistance index scores. RESULTS: In both groups (control [n = 27] and protein [n = 53]), there were significant (P ≤ .05) changes from baseline in weight loss (-6.2% and -7.2%), distance walked (+53.1 and +75.0 meters), and fasting plasma glucose (-7.5 and -6.2 mg/dL), respectively. At endpoint, protein group had significantly (P ≤ .05) lower triglycerides (-17.3 mg/dL), large very-low-density lipoprotein particle concentration (VLDL-P; -1.2 nmol/L), total low-density lipoprotein particle concentration (LDL-P; -67.8 nmol/L), small LDL-P (-59.4 nmol/L) and lipoprotein insulin resistance index (-5.9), whereas control group had significantly (P ≤ .05) lower GlycA (-13.1 µmol/L), total VLDL-P (-7.9 nmol/L), and small VLDL-P (-7.0 nmol/L). Differences between groups were observed for small VLDL-P (P = .02) and protein intake (P < .0001). CONCLUSIONS: These findings suggest that a hypocaloric diet with either traditional (0.8 g/kg BW/d) or higher protein (1.2 g/kg BW/d; predominantly from lean red meat) content improves risk markers of cardiovascular disease and type II diabetes in obese middle-aged and older adults. Both diets were also associated with improved physical function, and neither had an adverse impact on cardiometabolic outcomes.

Influence of Protein Intake, Race, and Age on Responses to a Weight-Reduction Intervention in Obese Women.

BACKGROUND: Women have higher rates of obesity than men and develop more pronounced functional deficits as a result. Yet, little is known about how obesity reduction affects their functional status, including whether their responses differ when protein intake is enhanced. OBJECTIVE: The aim of this study was to confirm the feasibility of delivery of a higher-protein (balanced at each meal) calorie-restricted diet in obese women and determine its efficacy for influencing function and retention of lean mass. METHOD: Obese community-dwelling women [n = 80; body mass index (in kg/m2), in means ± SDs: 37.8 ± 5.9; aged 45-78 y; 58.8% white] were enrolled in a weight-loss (-500 kcal/d) study and randomly assigned to either a Control-Weight-Loss (C-WL; 0.8 g protein/kg body weight) group or a High-Protein-Weight-Loss (HP-WL; 1.2 g protein/kg body weight; 30 g protein 3 times/d) group in a 1:2 allocation. Primary outcomes were function by 6-min walk test (6MWT) and lean mass by using the BodPod (Life Measurement, Inc.) at 0, 4, and 6 mo. RESULTS: Both groups reduced calorie intakes and body weights (P < 0.001), and the feasibility of the HP-WL intervention was confirmed. The 6MWT results improved (P < 0.01) at 4 mo in the HP-WL group and at 6 mo in both groups (P < 0.001). Both groups improved function by several other measures while slightly decreasing (P < 0.01) lean mass (-1.0 kg, C-WL; -0.6 kg, HP-WL). Weight loss was greater in white than in black women at both 4 mo (6.0 ± 3.6 compared with 3.7 ± 3.4 kg; P < 0.02) and 6 mo (7.2 ± 4.8 compared with 4.0 ± 4.7 kg; P < 0.04) and tended to be positively related to age (P < 0.06). CONCLUSIONS: A clinically important functional benefit of obesity reduction was confirmed in both study groups, with no significant group effect. Our findings of racial differences in response to the intervention and a potential influence of participant age lend support for further studies sufficiently powered to explore the interaction of race and age with functional responses to obesity reduction in women. This trial was registered at clinicaltrials.gov as NCT02033655.

Improved Function With Enhanced Protein Intake per Meal: A Pilot Study of Weight Reduction in Frail, Obese Older Adults.

BACKGROUND: Obesity is a significant cause of functional limitations in older adults; yet, concerns that weight reduction could diminish muscle along with fat mass have impeded progress toward an intervention. Meal-based enhancement of protein intake could protect function and/or lean mass but has not been studied during geriatric obesity reduction. METHODS: In this 6-month randomized controlled trial, 67 obese (body mass index ≥30kg/m(2)) older (≥60 years) adults with a Short Physical Performance Battery score of 4-10 were randomly assigned to a traditional (Control) weight loss regimen or one with higher protein intake (>30g) at each meal (Protein). All participants were prescribed a hypo-caloric diet, and weighed and provided dietary guidance weekly. Physical function (Short Physical Performance Battery) and lean mass (BOD POD), along with secondary measures, were assessed at 0, 3, and 6 months. RESULTS: At the 6-month endpoint, there was significant (p < .001) weight loss in both the Control (-7.5±6.2kg) and Protein (-8.7±7.4kg) groups. Both groups also improved function but the increase in the Protein (+2.4±1.7 units; p < .001) was greater than in the Control (+0.9±1.7 units; p < .01) group (p = .02). CONCLUSION: Obese, functionally limited older adults undergoing a 6-month weight loss intervention with a meal-based enhancement of protein quantity and quality lost similar amounts of weight but had greater functional improvements relative to the Control group. If confirmed, this dietary approach could have important implications for improving the functional status of this vulnerable population (ClinicalTrials.gov identifier: NCT01715753).

Exercise effects on lipids in persons with varying dietary patterns-does diet matter if they exercise? Responses in Studies of a Targeted Risk Reduction Intervention through Defined Exercise I.

BACKGROUND: The standard clinical approach for reducing cardiovascular disease risk due to dyslipidemia is to prescribe changes in diet and physical activity. The purpose of the current study was to determine if, across a range of dietary patterns, there were variable lipoprotein responses to an aerobic exercise training intervention. METHODS: Subjects were participants in the STRRIDE I, a supervised exercise program in sedentary, overweight subjects randomized to 6 months of inactivity or 1 of 3 aerobic exercise programs. To characterize diet patterns observed during the study, we calculated a modified z-score that included intakes of total fat, saturated fat, trans fatty acids, cholesterol, omega-3 fatty acids, and fiber as compared with the 2006 American Heart Association diet recommendations. Linear models were used to evaluate relationships between diet patterns and exercise effects on lipoproteins/lipids. RESULTS: Independent of diet, exercise had beneficial effects on low-density lipoprotein cholesterol particle number, low-density lipoprotein cholesterol size, high-density lipoprotein cholesterol, high-density lipoprotein cholesterol size, and triglycerides (P < .05 for all). However, having a diet pattern that closely adhered to American Heart Association recommendations was not related to changes in these or any other serum lipids or lipoproteins in any of the exercise groups. CONCLUSIONS: We found that even in sedentary individuals whose habitual diets vary in the extent of adherence to AHA dietary recommendations, a rigorous, supervised exercise intervention can achieve significant beneficial lipid effects.

Dietary carbohydrate intake and high-sensitivity C-reactive protein in at-risk women and men.

BACKGROUND: The quality and quantity of dietary carbohydrate intake, measured as dietary glycemic load (GL), are associated with a number of cardiovascular disease (CVD) risk factors and, in healthy young women, are related to increased high-sensitivity C-reactive protein (hsCRP) concentrations. Our objective was to determine if GL is related to hsCRP and other measures of CVD risk in a population of sedentary, overweight, dyslipidemic middle-aged women and men enrolled in an exercise intervention trial (STRRIDE). METHODS: This was a cross-sectional evaluation of the relationships between measures of dietary carbohydrate intake, calculated from food frequency questionnaire data, and CVD risk factors, including plasma hsCRP, measured in 171 subjects. RESULTS: After adjusting for energy intake, GL and other measures of carbohydrate intake were not independently related to hsCRP (P > .05 for all). In the analyses performed separately for each sex, only the quantity of carbohydrate intake was independently related to hsCRP (R2 = 0.28, P < .04), and this relationship was present for women but not for men. The strongest relationship identified between GL and any CVD risk factor was for cardiorespiratory fitness (R2 = 0.12, P < .02); an elevated GL was associated with a lower level of fitness in all subjects, and this relationship persisted even when the findings were adjusted for energy intake and sex (R2 = 0.48, P < .03). CONCLUSIONS: In middle-aged, sedentary, overweight to mildly obese, dyslipidemic individuals, consuming a diet with a low GL is associated with better cardiorespiratory fitness. Our findings suggest that the current literature relating carbohydrate intake and hsCRP should be viewed with skepticism, especially in the extension to at-risk populations that include men.