Proteomic profiling of human plasma extracellular vesicles identifies PF4 and C1R as novel biomarker in sarcopenia.

BACKGROUND: Sarcopenia, the gradual and generalized loss of muscle mass and function with ageing, is one of the major health problems in older adults, given its high prevalence and substantial socioeconomic implications. Despite the extensive efforts to reach consensus on definition and diagnostic tests and cut-offs for sarcopenia, there is an urgent and unmet need for non-invasive, specific and sensitive biomarkers for the disease. Extracellular vesicles (EVs) are present in different biofluids including plasma, whose cargo reflects cellular physiology. This work analysed EV proteome in sarcopenia and robust patients in the search for differentially contained proteins that can be used to diagnose the disease. METHODS: Plasma small EVs (sEVs) from a total of 29 robust controls (aged 73.4 ± 5.6 years; 11 men and 18 women) and 49 sarcopenic patients (aged 82.3 ± 5.4 years; 15 men and 34 women) aged 65 years and older were isolated and their cargo was analysed by proteomics. Proteins whose concentration in sEVs was different between sarcopenic and robust patients were further validated using ELISA. The concentration of these candidates was correlated to the EWGSOP2 sarcopenia tests for low muscle strength and low physical performance, and receiver operating characteristic (ROC) curve analyses were carried out to evaluate their diagnostic power, sensitivity and specificity. RESULTS: Proteomic analysis identified 157 sEVs proteins in both sarcopenic and robust samples. Among them, 48 proteins had never been reported in the ExoCarta nor Vesiclepedia databases. Statistical analysis revealed eight proteins whose concentration was significantly different between groups: PF4 (log2 FC = 4.806), OIT3 (log2 FC = -1.161), MMRN1 (log2 FC = -1.982), MASP1 (log2 FC = -0.627), C1R (log2 FC = 1.830), SVEP1 (log2 FC = 1.295), VCAN (FC = 0.937) and SPTB (log2 FC = 1.243). Among them, platelet factor 4 (PF4) showed the lowest concentration while Complement C1r subcomponent (C1R) increased the most in sarcopenic patients, being these results confirmed by ELISA (P = 1.07E-09 and P = 0.001287, respectively). The concentrations of candidate proteins significantly correlated with EWGSOP2 tests currently used. ROC curve analysis showed an area under the curve of 0.8921 and 0.7476 for PF4 and C1R, respectively. Choosing the optimal for PF4, 80% sensitivity and 85.71% specificity was reached while the optimal cut-off value of C1R would allow sarcopenia diagnosis with 75% sensitivity and 66.67% specificity. CONCLUSIONS: Our results support the determination of EV PF4 and C1R as plasma diagnostic biomarkers in sarcopenia and open the door to investigate the role of the content of these vesicles in the pathogeny of the disease.

Fitness vs Fatness as Determinants of Survival in Noninstitutionalized Older Adults: The EXERNET Multicenter Study.

BACKGROUND: Physical fitness and body composition are important health indicators; nevertheless, their combined pattern interrelationships and their association with mortality are poorly investigated. METHODS: This longitudinal study is part of the Spanish EXERNET-Elder project. Person-months of follow-up were calculated from the interview date, performed between June 2008 and November 2009, until the date of death or censoring on March 2018 (whichever came first). In order to be included, participants had to fulfill the following criteria: (a) be older than 65 years, (b) live independently at home, (c) not suffer dementia and/or cancer, and (d) have a body mass index above 18.5. Body fat and weight were assessed by a bioelectrical impedance analyzer. Fitness was measured with the Senior Fitness and the one-leg static balance tests. The Spanish Death Index was consulted for the death's identification. Cluster analysis was performed to identify Fat-Fit patterns and traditional cut-points and percentiles to create the Fat-Fit groups. Cox proportional hazards regression models were used to calculate the hazard ratios (HRs) of death in clustered Fat-Fit patterns and in traditional Fat-Fit groups. RESULTS: A total of 2299 older adults (76.8% of women) were included with a baseline mean age of 71.9 ± 5.2 years. A total of 196 deaths (8.7% of the sample) were identified during the 8 years of follow-up. Four clustered Fat-Fit patterns (Low fat-Fit, Medium fat-Fit, High fat-Unfit, and Low fat-Unfit) and 9 traditional Fat-Fit groups emerged. Using the Low fat-Fit pattern as the reference, significantly increased mortality was noted in High fat-Unfit (HR: 1.68, CI: 1.06-2.66) and Low fat-Unfit (HR: 2.01, CI: 1.28-3.16) groups. All the traditional Fit groups showed lower mortality risk when compared to the reference group (obese-unfit group). CONCLUSION: Physical fitness is a determinant factor in terms of survival in community-dwelling older adults, independently of adiposity levels.

Fat-Fit Patterns, Drug Consumption, and Polypharmacy in Older Adults: The EXERNET Multi-Center Study.

BACKGROUND: Physical fitness levels and the amount of accumulated adipose tissue (fatness) relate to current and future individuals' heath status. Nevertheless, the interrelationships of their combined patterns with polypharmacy and the types of medications consumed have not been sufficiently investigated. METHODS: This cross-sectional study was carried out in six Spanish regions between 2008 and 2009 with a sample of older community-dwelling adults (≥65 years old) without dementia or cancer. Fitness was measured with one-leg balance and senior fitness tests, as well as by measuring weight and fat mass with a bioelectrical impedance analyzer. Polypharmacy was defined as the use of five or more medications. An analysis of variance was performed for comparisons between the physical fitness and fatness patterns and the medication consumed. RESULTS: A total of 1709 elders were included in the study (72.1 ± 5.2 years). The two unfit patterns were those with the highest drug consumption. The High-Fat-Unfit pattern was the one that had the most significant consumption and had the highest percentage of polymedicated subjects. The Low-Fat-Fit pattern had a significantly lower percentage of people that did not consume any medications. The highest percentages of drug consumption in 7 of the 10 groups that were included were concentrated in the two unfit patterns. CONCLUSIONS: This study highlights the importance of fitness in older adults, as it is at least as important as the avoidance of accumulation of excess fat with respect to the consumption of a smaller number of medicines.

'Fat but powerful' paradox: association of muscle power and adiposity markers with all-cause mortality in older adults from the EXERNET multicentre study.

OBJECTIVES: To assess the influence of muscle power and adiposity on all-cause mortality risk and to evaluate the 'fat but powerful' (F+P) (or 'fat but fit') paradox in older adults. METHODS: A total of 2563 older adults (65‒91 years old) from the EXERNET multicentre study were included. Adiposity (body mass index (BMI), waist circumference, body fat percentage (BF%) and fat index), allometric and relative power (sit-to-stand muscle power test) and various covariates (age, sex, hypertension, smoking status and walking and sitting times per day) were registered at baseline. All-cause mortality was recorded during a median follow-up of 8.9 years. Participants were classified into four groups: lean and powerful (L+P), F+P, lean but weak and fat and weak (F+W). Cox proportional hazard regression models and adjusted HRs were calculated. RESULTS: According to BMI and waist circumference, all-cause mortality risk was reduced in the F+P (HR=0.55 and 0.63, p=0.044 and 0.049, respectively) and L+P (HR=0.57 and 0.58, p=0.043 and 0.025, respectively) groups. According to BF%, all-cause mortality decreased in the L+P group (HR=0.53; p=0.021), and a trend for a reduction was reported in the F+P group (HR=0.57; p=0.060). According to fat index, a survival benefit was only noted in the L+P group (HR=0.50; p=0.049). Higher levels of relative power reduced all-cause mortality risk among older people (HR=0.63 and 0.53, p=0.006 and 0.011, respectively). CONCLUSION: Powerful older people exhibited a reduced 9-year all-cause mortality regardless of BMI, waist circumference and BF%. Obesity according to fat index blunted the survival benefits of being powerful.

Effects of whole-body vibration training on bone density and turnover markers in adolescent swimmers.

Background Whole-body vibration training has recently been proposed as a complementary training modality to improve the bone health of adolescent swimmers. However, there is no longitudinal study regarding the effects of this training combination on bone metabolism. Therefore, the main goal was to analyze the effects of swimming and vibration training on bone turnover markers during adolescence. Methods The present study included 68 adolescent swimmers and 41 normoactive controls (CON). Swimmers were randomly selected to either continue with their regular swimming training (SWI) or participate in an additional vibration protocol (VIB). Anthropometric measurements and serum level determinations of osteocalcin (OC), procollagen type 1 N-terminal propeptide (P1NP) and C-terminal telopeptide crosslaps (CTX) were performed before and after the 6-month intervention. Results Statistically significant group by time interactions were found for both bone formation markers. VIB showed a decrease over time in OC (baseline: 101.4 µg/mL, follow-up: 82.8 µg/mL, p < 0.05) and P1NP (baseline: 528.4 µg/mL, follow-up: 389.0 µg/mL, p < 0.05) and SWI had analogous reductions in P1NP (baseline: 685.8 µg/mL, follow-up: 542.0 µg/mL, p < 0.05), whereas CON experienced an increase in OC levels (baseline: 94.4 µg/mL, follow-up: 103.4 µg/mL, p < 0.05). After stratifying the sample according to the pubertal status, similar interactions were observed. Conclusions The combination of swimming training and this particular vibration protocol led to a decrease in bone formation markers, especially during early puberty. Whole-body vibration might not induce an osteogenic stimulus in adolescent swimmers.

Influences of physical fitness on bone mass in women with fibromyalgia.

The aim of this study was to provide information about the relationship of bone mineral content (BMC) and density (BMD) with some physical-fitness-related variables in a sample of women with fibromyalgia (FM) and age-matched women without FM. Twenty-eight women clinically diagnosed with FM (age 51.1 ± 8.4 yr, M ± SD) and 22 age-matched controls participated in the study. Whole-body BMC and BMD, lean mass, handgrip strength, quadriceps strength, and cardiovascular fitness were measured in all participants. The association between physical-fitness variables and bone-related variables was tested by linear regression controlling for body weight as a possible confounder. There were no differences in BMC or BMD between groups. Women with FM had lower values of handgrip strength, quadriceps strength, and VO2peak than the control group. Handgrip strength and aerobic capacity were associated with BMC and BMD and quadriceps strength was associated with BMD in women with FM; however, only VO2peak was associated with BMC in the group of women without FM. Bone mass of women with FM may be more susceptible to changes in physical fitness than that of the women without fibromyalgia.

[Physical fitness evolution in octogenarian population and its relationship with a sedentary lifestyle]. / Evolución de los niveles de condición física en población octogenaria y su relación con un estilo de vida sedentario.

OBJECTIVE: The aim of this study was to determine the changes in physical fitness over two years of following up in octogenarian people and to check whether a sedentary lifestyle modify these variations. METHODS: Physical fitness of 182 subject (48 men, 134 women) with a mean age of 82,3 ± 2,3 years were evaluated using 8 different tests. A repeated measures analysis was carried out to see the differences between the two evaluation periods and to see the physical fitness differences between sedentary people (sit ≥ 4 hours/day) and non sedentary people (sit < 4 hours/day). RESULTS: Between the two evaluation periods, we found a significant decrease in the agility test (p < 0.05), walking speed (p < 0.01) and endurance (p < 0.01). In relation to the subjects who spent sitting 4 hours/day there was a decrease in the walking speed test between the two evaluations (p < 0.05). Moreover, there was a decrease of walking speed and endurance between the two evaluation periods in both sedentary and nonsedentary people (p < 0.05). CONCLUSION: In two years of following up, there are adverse changes in the level of physical fitness in octogenarians. Long periods of sitting time may translate into a loss of agility. Walking speed and endurance seem to be the components of physical fitness more affected by the ageing process in this population; and this loss is not determined by the hours of sitting per day.

Objetivos: Determinar los cambios que se producen en la condición física a lo largo de dos años de seguimiento en octogenarios y comprobar si un estilo de vida sedentario modifica estas variaciones. Metodología: La condición física de 182 sujetos (48 hombres, 134 mujeres) con una media de edad de 82,3 ± 2,3 años se evaluó mediante 8 test (adaptados de las baterías "Senior Fitness Test" y "Eurofit"). Se realizó un análisis de medidas repetidas entre los dos periodos de evaluación y para ver las diferencias de condición física entre los sujetos considerados sedentarios (permanecían sentados ≥4 horas/día) y no sedentarios (permanecían sentados < 4 horas/día) Resultados: Entre las dos evaluaciones encontramos un descenso significativo en los test de agilidad (p < 0,05), velocidad (p < 0,01) y resistencia (p < 0,01) y un ligero aumento de la fuerza de brazos en los sujetos que permanecían sentados < 4 horas/día (p < 0,05). Aquellos que permanecían sentados > 4 horas/día sufrieron un descenso de la agilidad (p < 0,05). Se produjo una pérdida de velocidad y resistencia tanto en sedentarios como en no sedentarios (p < 0,05). Conclusión: En dos años de seguimiento, se producen cambios negativos en el nivel de condición física en personas octogenarias. Periodos prolongados de sedestación se traducen en una pérdida de agilidad. La velocidad de la marcha y resistencia parecen ser los componentes de la condición física más afectados por el proceso de envejecimiento en este grupo poblacional y su pérdida no viene determinada por las horas diarias de sedestación.

An optimized histochemical method to assess skeletal muscle glycogen and lipid stores reveals two metabolically distinct populations of type I muscle fibers.

Skeletal muscle energy metabolism has been a research focus of physiologists for more than a century. Yet, how the use of intramuscular carbohydrate and lipid energy stores are coordinated during different types of exercise remains a subject of debate. Controversy arises from contradicting data from numerous studies, which used different methodological approaches. Here we review the "pros and cons" of previously used histochemical methods and describe an optimized method to ensure the preservation and specificity of detection of both intramyocellular carbohydrate and lipid stores. For optimal preservation of muscle energy stores, air drying cryosections or cycles of freezing-thawing need to be avoided. Furthermore, optimization of the imaging settings in order to specifically image intracellular lipid droplets stained with oil red O or Bodipy-493/503 is shown. When co-staining lipid droplets with associated proteins, Bodipy-493/503 should be the dye of choice, since oil red O creates precipitates on the lipid droplets blocking the light. In order to increase the specificity of glycogen stain, an antibody against glycogen is used. The resulting method reveals the existence of two metabolically distinct myosin heavy chain I expressing fibers: I-1 fibers have a smaller crossectional area, a higher density of lipid droplets, and a tendency to lower glycogen content compared to I-2 fibers. Type I-2 fibers have similar lipid content than IIA. Exhaustive exercise lead to glycogen depletion in type IIA and IIX fibers, a reduction in lipid droplets density in both type I-1 and I-2 fibers, and a decrease in the size of lipid droplets exclusively in type I-1 fibers.

[Sarcopenic obesity and physical fitness in octogenarians: the multi-center EXERNET Project]. / Obesidad sarcopénica y condición física en octogenarios; proyecto multi-céntrico EXERNET.

OBJECTIVE: The aim of this study was to analyze the usefulness of different fitness test to detect the risk of sarcopenic obesity (SO) in octogenarian people. METHODS: 306 subjects (76 men, 230 women) with a mean age of 82.5 ± 2.3 years from the Multi-center EXERNET Project sample fulfilled the inclusion criteria. Body composition was assessed in all subjects by bioelectrical impedance. Four groups were created based on the percentage of fat mass and muscle mass: 1) normal, 2) high fat mass, 3) low muscle mass and 4) SO. Physical fitness was assessed using 8 different tests modified from the batteries "Senior Fitness Test" and Eurofit (EXERNET battery). The risk of suffering SO depending on the fitness level was studied by logistic regression. RESULTS: Among the studied physical fitness tests, those that better predicted the risk of SO were leg strength, arm strength, agility, walking speed and balance in men; 95% CI [(0.606-0.957) (0.496-0.882), (0.020-2.014), (0.17-1.39), (0.913-1.002), all p < 0.05, except balance test (p = 0.07)] and balance test and agility in women; (95% CI [(0.928-1.002) (0.983-1.408), (both p = 0.07)]. CONCLUSION: Adequate levels of physical fitness are associated with a lower risk of SO. Some easy fitness tests seem to be useful for the detection of SO in those cases where the body-composition required methods for diagnosis are not available.

Objetivo: El objetivo de este estudio fue analizar la utilidad de diferentes test de condición física para detectar el riesgo de sufrir obesidad sarcopénica (OS) en octogenarios. Métodos: 306 sujetos (76 hombres, 230 mujeres) con una media de edad de 82,5±2,3 años, del total de la muestra del Proyecto Multi-céntrico EXERNET cumplieron con los criterios de inclusión. La composición corporal se evaluó en todos los sujetos mediante impedancia bioeléctrica. Se crearon 4 grupos basados en el porcentaje de masa grasa y masa muscular: 1) normal, 2) elevada masa grasa, 3) baja masa muscular y 4) OS. La condición física fue evaluada utilizando 8 test diferentes adaptados de las baterías "Senior Fitness Test" y "Eurofit" (batería EXERNET). La probabilidad de padecer OS en función del nivel de condición física se estudió mediante regresión logística. Resultados: Entre las pruebas de condición física estudiadas, los test que mejor predecían el riesgo de sufrir OS fueron la falta de fuerza de piernas, fuerza de brazos, agilidad, velocidad y equilibrio en hombres; 95% CI [(0,606-0,957); (0,496-0,882); (1,116-3,636); (1,060-1,825); (0,913-1,002); todos p.