Elena Volpi

Evidence-Based Recommendations for Optimal Dietary Protein Intake in Older People: A Position Paper From the PROT-AGE Study Group

New evidence shows that older adults need more dietary protein than do younger adults to support good health, promote recovery from illness, and maintain functionality. Older people need to make up for age-related changes in protein metabolism, such as high splanchnic extraction and declining anabolic responses to ingested protein. They also need more protein to offset inflammatory and catabolic conditions associated with chronic and acute diseases that occur commonly with aging. With the goal of developing updated, evidence-based recommendations for optimal protein intake by older people, the European Union Geriatric Medicine Society (EUGMS), in cooperation with other scientific organizations, appointed an international study group to review dietary protein needs with aging (PROT-AGE Study Group). To help older people (>65 years) maintain and regain lean body mass and function, the PROT-AGE study group recommends average daily intake at least in the range of 1.0 to 1.2 g protein per kilogram of body weight per day. Both endurance- and resistance-type exercises are recommended at individualized levels that are safe and tolerated, and higher protein intake (ie, ≥ 1.2 g/kg body weight/d) is advised for those who are exercising and otherwise active. Most older adults who have acute or chronic diseases need even more dietary protein (ie, 1.2-1.5 g/kg body weight/d). Older people with severe kidney disease (ie, estimated GFR <30 mL/min/1.73 m(2)), but who are not on dialysis, are an exception to this rule; these individuals may need to limit protein intake. Protein quality, timing of ingestion, and intake of other nutritional supplements may be relevant, but evidence is not yet sufficient to support specific recommendations. Older people are vulnerable to losses in physical function capacity, and such losses predict loss of independence, falls, and even mortality. Thus, future studies aimed at pinpointing optimal protein intake in specific populations of older people need to include measures of physical function.

Resistance exercise increases AMPK activity and reduces 4E‐BP1 phosphorylation and protein synthesis in human skeletal muscle

Resistance exercise is a potent stimulator of muscle protein synthesis and muscle cell growth, with the increase in protein synthesis being detected within 2–3 h post‐exercise and remaining elevated for up to 48 h. However, during exercise, muscle protein synthesis is inhibited. An increase in AMP‐activated protein kinase (AMPK) activity has recently been shown to decrease mammalian target of rapamycin (mTOR) signalling to key regulators of translation initiation. We hypothesized that the cellular mechanism for the inhibition of muscle protein synthesis during an acute bout of resistance exercise in humans would be associated with an activation of AMPK and an inhibition of downstream components of the mTOR pathway (4E‐BP1 and S6K1). We studied 11 subjects (seven men, four women) before, during, and for 2 h following a bout of resistance exercise. Muscle biopsy specimens were collected at each time point from the vastus lateralis. We utilized immunoprecipitation and immunoblotting methods to measure muscle AMPKα2 activity, and mTOR‐associated upstream and downstream signalling proteins, and stable isotope techniques to measure muscle fractional protein synthetic rate (FSR). AMPKα2 activity (pmol min−1 (mg protein)−1) at baseline was 1.7 ± 0.3, increased immediately post‐exercise (3.0 ± 0.6), and remained elevated at 1 h post‐exercise (P < 0.05). Muscle FSR decreased during exercise and was significantly increased at 1 and 2 h post‐exercise (P < 0.05). Phosphorylation of 4E‐BP1 at Thr37/46 was significantly reduced immediately post‐exercise (P < 0.05). We conclude that AMPK activation and a reduced phosphorylation of 4E‐BP1 may contribute to the inhibition of muscle protein synthesis during resistance exercise. However, by 1–2 h post‐exercise, muscle protein synthesis increased in association with an activation of protein kinase B, mTOR, S6K1 and eEF2.

Skeletal muscle protein anabolic response to resistance exercise and essential amino acids is delayed with aging

Skeletal muscle loss during aging leads to an increased risk of falls, fractures, and eventually loss of independence. Resistance exercise is a useful intervention to prevent sarcopenia; however, the muscle protein synthesis (MPS) response to resistance exercise is less in elderly compared with young subjects. On the other hand, essential amino acids (EAA) increase MPS equally in both young and old subjects when sufficient EAA is ingested. We hypothesized that EAA ingestion following a bout of resistance exercise would stimulate anabolic signaling and MPS similarly between young and old men. Each subject ingested 20 g of EAA 1 h following leg resistance exercise. Muscle biopsies were obtained before and 1, 3, and 6 h after exercise to measure the rate of MPS and signaling pathways that regulate translation initiation. MPS increased early in young (1-3 h postexercise) and later in old (3-6 h postexercise). At 1 h postexercise, ERK1/2 MNK1 phosphorylation increased and eIF2alpha phosphorylation decreased only in the young. mTOR signaling (mTOR, S6K1, 4E-BP1, eEF2) was similar between groups at all time points, but MNK1 phosphorylation was lower at 3 h and AMP-activated protein kinase-alpha (AMPKalpha) phosphorylation was higher in old 1-3 h postexercise. We conclude that the acute MPS response after resistance exercise and EAA ingestion is similar between young and old men; however, the response is delayed with aging. Unresponsive ERK1/2 signaling and AMPK activation in old muscle may be playing a role in the delayed activation of MPS. Notwithstanding, the combination of resistance exercise and EAA ingestion should be a useful strategy to combat sarcopenia.

Rapamycin administration in humans blocks the contraction-induced increase in skeletal muscle protein synthesis

Muscle protein synthesis and mTORC1 signalling are concurrently stimulated following muscle contraction in humans. In an effort to determine whether mTORC1 signalling is essential for regulating muscle protein synthesis in humans, we treated subjects with a potent mTORC1 inhibitor (rapamycin) prior to performing a series of high‐intensity muscle contractions. Here we show that rapamycin treatment blocks the early (1–2 h) acute contraction‐induced increase (∼40%) in human muscle protein synthesis. In addition, several downstream components of the mTORC1 signalling pathway were also blunted or blocked by rapamycin. For instance, S6K1 phosphorylation (Thr421/Ser424) was increased post‐exercise 6‐fold in the control group while being unchanged with rapamycin treatment. Furthermore, eEF2 phosphorylation (Thr56) was reduced by ∼25% post‐exercise in the control group but phosphorylation following rapamycin treatment was unaltered, indicating that translation elongation was inhibited. Rapamycin administration prior to exercise also reduced the ability of raptor to associate with mTORC1 during post‐exercise recovery. Surprisingly, rapamycin treatment prior to resistance exercise completely blocked the contraction‐induced increase in the phosphorylation of ERK1/2 (Thr202/Tyr204) and blunted the increase in MNK1 (Thr197/202) phosphorylation. However, the phosphorylation of a known target of MNK1, eIF4E (Ser208), was similar in both groups (P > 0.05) which is consistent with the notion that rapamycin does not directly inhibit MAPK signalling. We conclude that mTORC1 signalling is, in part, playing a key role in regulating the contraction‐induced stimulation of muscle protein synthesis in humans, while dual activation of mTORC1 and ERK1/2 stimulation may be required for full stimulation of human skeletal muscle protein synthesis.

Brown Adipose Tissue Improves Whole-Body Glucose Homeostasis and Insulin Sensitivity in Humans

Brown adipose tissue (BAT) has attracted scientific interest as an antidiabetic tissue owing to its ability to dissipate energy as heat. Despite a plethora of data concerning the role of BAT in glucose metabolism in rodents, the role of BAT (if any) in glucose metabolism in humans remains unclear. To investigate whether BAT activation alters whole-body glucose homeostasis and insulin sensitivity in humans, we studied seven BAT-positive (BAT+) men and five BAT-negative (BAT−) men under thermoneutral conditions and after prolonged (5–8 h) cold exposure (CE). The two groups were similar in age, BMI, and adiposity. CE significantly increased resting energy expenditure, whole-body glucose disposal, plasma glucose oxidation, and insulin sensitivity in the BAT+ group only. These results demonstrate a physiologically significant role of BAT in whole-body energy expenditure, glucose homeostasis, and insulin sensitivity in humans, and support the notion that BAT may function as an antidiabetic tissue in humans.

Insulin resistance of muscle protein metabolism in aging.

A reduced response of older skeletal muscle to anabolic stimuli may contribute to the development of sarcopenia. We hypothesized that muscle proteins are resistant to the anabolic action of insulin in the elderly. We examined the effects of hyperinsulinemia on muscle protein metabolism in young (25±2 year) and older (68±1 year) healthy subjects using stable isotope tracer techniques. Leg blood flow was higher in the young at baseline and increased during hyperinsulinemia, whereas it did not change in the elderly. Glucose concentrations and muscle uptake were not different between groups at baseline and during hyperinsulinemia. Leg phenylalanine net balance was not different at baseline and significantly increased in both groups with hyperinsulinemia (P<0.05) but to a greater extent in the young (P<0.05). Muscle protein synthesis increased only in the young during hyperinsulinemia. Muscle protein breakdown did not significantly change in either group, although it tended to decrease in the elderly. Changes in muscle protein synthesis were correlated with changes in leg amino acid delivery (R=0.89; P=0.0001) and blood flow (R=0.90; P<0.0001). In conclusion, skeletal muscle protein synthesis is resistant to the anabolic action of insulin in older subjects, which may be an important contributor to the development of sarcopenia.

Aging impairs contraction-induced human skeletal muscle mTORC1 signaling and protein synthesis

BackgroundSarcopenia, the loss of skeletal muscle mass during aging, increases the risk for falls and dependency. Resistance exercise (RE) training is an effective treatment to improve muscle mass and strength in older adults, but aging is associated with a smaller amount of training-induced hypertrophy. This may be due in part to an inability to stimulate muscle-protein synthesis (MPS) after an acute bout of RE. We hypothesized that older adults would have impaired mammalian target of rapamycin complex (mTORC)1 signaling and MPS response compared with young adults after acute RE.MethodsWe measured intracellular signaling and MPS in 16 older (mean 70 ± 2 years) and 16 younger (27 ± 2 years) subjects. Muscle biopsies were sampled at baseline and at 3, 6 and 24 hr after exercise. Phosphorylation of regulatory signaling proteins and MPS were determined on successive muscle biopsies by immunoblotting and stable isotopic tracer techniques, respectively.ResultsIncreased phosphorylation was seen only in the younger group (P< 0.05) for several key signaling proteins after exercise, including mammalian target of rapamycin (mTOR), ribosomal S6 kinase (S6K)1, eukaryotic initiation factor 4E-binding protein (4E-BP)1 and extracellular signal-regulated kinase (ERK)1/2, with no changes seen in the older group (P > 0.05). After exercise, MPS increased from baseline only in the younger group (P< 0.05), with MPS being significantly greater than that in the older group (P < 0.05).ConclusionsWe conclude that aging impairs contraction-induced human skeletal muscle mTORC1 signaling and protein synthesis. These age-related differences may contribute to the blunted hypertrophic response seen after resistance-exercise training in older adults, and highlight the mTORC1 pathway as a key therapeutic target to prevent sarcopenia.

Is the Optimal Level of Protein Intake for Older Adults Greater Than the Recommended Dietary Allowance

BACKGROUND Protein is a macronutrient essential for growth, muscle function, immunity and overall tissue homeostasis. Suboptimal protein intake can significantly impact physical function and overall health in older adults. METHODS This article reviews the literature on the recommendations for protein intake in older adults in light of the new evidence linking protein intake with sarcopenia and physical function. Challenges and opportunities for optimal protein nutrition in older persons are discussed. RESULTS Recent metabolic and epidemiological studies suggest that the current recommendations of protein intake may not be adequate for maintenance of physical function and optimal health in older adults. Methodological limitations and novel concepts in protein nutrition are also discussed. CONCLUSION We conclude that new research and novel research methodologies are necessary to establish the protein needs and optimal patterns of protein intake for older persons.

Aging and microRNA expression in human skeletal muscle: a microarray and bioinformatics analysis

A common characteristic of aging is loss of skeletal muscle (sarcopenia), which can lead to falls and fractures. MicroRNAs (miRNAs) are novel posttranscriptional modulators of gene expression with potential roles as regulators of skeletal muscle mass and function. The purpose of this study was to profile miRNA expression patterns in aging human skeletal muscle with a miRNA array followed by in-depth functional and network analysis. Muscle biopsy samples from 36 men [young: 31 ± 2 (n = 19); older: 73 ± 3 (n = 17)] were 1) analyzed for expression of miRNAs with a miRNA array, 2) validated with TaqMan quantitative real-time PCR assays, and 3) identified (and later validated) for potential gene targets with the bioinformatics knowledge base software Ingenuity Pathways Analysis. Eighteen miRNAs were differentially expressed in older humans (P < 0.05 and >500 expression level). Let-7 family members Let-7b and Let-7e were significantly elevated and further validated in older subjects (P < 0.05). Functional and network analysis from Ingenuity determined that gene targets of the Let-7s were associated with molecular networks involved in cell cycle control such as cellular proliferation and differentiation. We confirmed with real-time PCR that mRNA expression of cell cycle regulators CDK6, CDC25A, and CDC34 were downregulated in older compared with young subjects (P < 0.05). In addition, PAX7 mRNA expression was lower in older subjects (P < 0.05). These data suggest that aging is characterized by a higher expression of Let-7 family members that may downregulate genes related to cellular proliferation. We propose that higher Let-7 expression may be an indicator of impaired cell cycle function possibly contributing to reduced muscle cell renewal and regeneration in older human muscle.

Human Muscle Gene Expression Following Resistance Exercise and Blood Flow Restriction

INTRODUCTION Blood flow restriction in combination with low-intensity resistance exercise (REFR) increases skeletal muscle size to a similar extent as compared with traditional high-intensity resistance exercise training. However, there are limited data describing the molecular adaptations that occur after REFR. PURPOSE To determine whether hypoxia inducible factor-1 alpha (HIF-1alpha) and REDD1 mRNA are expressed differently in REFR compared with low-intensity resistance exercise with no blood flow restriction (CONTROL). Secondly, to determine whether low-intensity resistance exercise is able to induce changes in mRNA expression of several anabolic and catabolic genes as typically seen with high-intensity resistance exercise. METHODS Six subjects were studied at baseline and 3 h after a bout of leg resistance exercise (20% 1RM) in REFR and CONTROL subjects. Each subject participated in both groups, with 3 wk separating each visit. Muscle biopsy samples were analyzed for mRNA expression, using qRT-PCR. RESULT Our primary finding was that there were no differences between CONTROL and REFR for any of the selected genes at 3 h after exercise (P > 0.05). However, low-intensity resistance exercise increased HIF-1alpha, p21, MyoD, and muscle RING finger 1 (MuRF1) mRNA expression and decreased REDD1 and myostatin mRNA expression in both groups (P < 0.05). CONCLUSION Low-intensity resistance exercise can alter skeletal muscle mRNA expression of several genes associated with muscle growth and remodeling, such as REDD1, HIF-1alpha, MyoD, MuRF1, and myostatin. Further, the results from REFR and CONTROL were similar, indicating that the changes in early postexercise gene expression were attributable to the low-intensity resistance exercise bout, and not blood flow restriction.