Inflammaging and Muscle Protein Metabolism

Study Overview
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Brief Summary
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The development of a low-grade, chronic, systemic inflammation observed in the elderly (inflammaing) has been associated with increased risk for skeletal muscle wasting, strength loss and functional impairments. According to studies performed in animals and cell cultures increased concentrations of pro-inflammatory cytokines such as IL-6 and TNF-α as well as increased levels of hs-CRP lead to elevated protein degradation through proteasome activation and reduced muscle protein synthesis (MPS) via downregulation of the Akt-mTOR signaling pathway. However, evidence regarding the effects of inflammaging on skeletal muscle mass in humans is lacking. Thus, the present study will compare proteasome activation and the protein synthetic response in the fasted and postprandial period between older adults with increased systemic inflammation and their healthy control counterparts.

Detailed Description
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A total number of > 60 male, older adults aged 63-73 years will be initially screened for systemic levels of hs-CRP and IL-6. Of these, 24 individuals that will comply with the study criteria will be allocated to either a High (IL6: ≥ 1.7 pg/ml; hs-CRP: > 1.0 mg/L) (n=12) or a Low (IL6: < 1.7 pg/ml; hs-CRP: < 1.0 mg/L) (n=12) systemic inflammation group. Approximately 2 weeks before the experimental trial, anthropometry, resting metabolic rate (RMR), body composition (with DXA), sarcopenia status, functional capacity and the knee-extension one repetition maximum (1RM) will be assessed in individuals from both groups. In addition, levels of habitual physical activity will be assessed using accelerometry (over a 7-day period) and daily dietary intake will be monitored through 7-day diet recalls in all participants. 1 week before the experimental day an oral glucose tolerance test (OGTT) will be also performed over a 2-hour period, with blood sampling every 15min during the first hour and every 30min during the second hour. The day before the experimental trial, participants will consume 150ml of D2O 70% atom as a bolus. In the experimental day, participants will arrive at the laboratory after an overnight fast and a baseline blood sample and a muscle biopsy from vastus lateralis muscle will be collected. Immediately after, participants will perform 8 sets with 10 repetitions at 80% of 1RM and 2 min rest between each set, on a knee-extension machine. After exercise, they will ingest 0.4 g whey protein isolate/kg body weight as single bolus and then they will remain in a sitting position over a 3-hour period. Blood samples will be collected every 30min during the 3-hour postprandial period while a second muscle biopsy will be obtained at 3h.

Official Title
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Effects of Low-grade Systemic Inflammation on Muscle Protein Synthesis and Breakdown in the Aged Skeletal Muscle.

Conditions
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Inflammaging, Sarcopenia

Intervention / Treatment
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* Dietary Supplement: Instantized Whey Protein Isolate


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Non-smokers. BMI ≥18.5 & BMI ≤ 35 kg/m2. Moderately active but with no regular participation in heavy resistance exercise within the last 6 months. Absence of chronic disease (i.e. cancer, metabolic, cardiac, or neurological diseases). Free and independently living. Exclusion Criteria: Organ failure (unstable, renal, respiratory, liver). Chronic use of corticosteroid medication. Recent use of antibiotics. Presence of frailty. Body weight variation over the past 6mo > 10% or weight loss of more than 3kg in the last 3 months. Use of anti-inflammatory or lipid-lowering medication (i.e., statins). Use of medication interacting with muscle metabolism.

Ages Eligible for Study
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Minimum Age: 63 Years
Maximum Age: 75 Years

Sexes Eligible for Study
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Male

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Non-Randomized
Intervention Model: Parallel Assignment
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: High systemic inflammation<br>Individuals assigned in the high systemic inflammation group will be characterized by IL6: ≥ 1.7 pg/ml and hs-CRP: > 2.0 mg/L. | Dietary Supplement: Instantized Whey Protein Isolate<br>* 0,4 g of whey protein isolate/kg body weight will be ingested as a bolus of 250 ml immediately after the resistance exercise bout.<br>|
| Active Comparator: Low systemic inflammation<br>Individuals assigned in the high systemic inflammation group will be characterized by IL6: < 1.7 pg/ml and hs-CRP: < 1.0 mg/L. | Dietary Supplement: Instantized Whey Protein Isolate<br>* 0,4 g of whey protein isolate/kg body weight will be ingested as a bolus of 250 ml immediately after the resistance exercise bout.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Systemic inflammation | Levels of systemic inflammation will be assessed by measuring serum levels of hs-CRP, IL-6 and TNF-α. | At baseline. | 
| Change in muscle protein synthesis (MPS) | Using deuterium oxide (D2O) 70% atom administration. Individuals will consume a single bolus of 150ml D20 the day before the clinical trial and muscle biopsy samples, collected before and after the exercise bout and protein ingestion, will be analyzed for isotopic measurement using GC-P-IRMS. | At baseline and 180 min following protein ingestion. | 
| Change in intracellular signaling proteins in muscle | Phosphorylation levels of Akt, mammalian target of rapamycin (mTOR), p70S6K and ribosomal protein S6 (rpS6) will be measure using western blotting. | At baseline and 180 min following protein ingestion. | 
| Change in proteasome activities in muscle | Chymotrypsin-like (CT-L), caspase-like (C-L) and trypsin-like (T-L) proteasome activities will be assayed with hydrolysis of the fluorogenic peptide LLVY-AMC, LLE-AMC and LSTR-AMC, respectively. | At baseline and 180 min following protein ingestion. | 
| Change in protein expression level of proteasome subunits | Immunoblot analysis will be used to detect protein expression levels of proteasome (β5, β2 and β1) and immunoproteasome (β5i, β2i and β1i) subunits. | At baseline and 180 min following protein ingestion. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Resting metabolic rate (RMR) | RMR will be assessed after an overnight fast with participants in a supine position following a 15-min stabilization period by taking 30 consecutive 1-min VO2/CO2 measurements using a portable open-circuit indirect calorimeter with a ventilated hood system following a standard calibration protocol. | At baseline. | 
| Physical activity | Level of habitual physical activity will be assessed using accelerometry (ActiGraph GT3X-BT accelerometer). | Over a 7-day period at baseline. | 
| Dietary intake | Daily dietary intake will be assessed using 7-day diet recalls. | Over a 7-day period at baseline. | 
| Reduced glutathione in blood | Concentration of reduced glutathione will be measured in red blood cells | At baseline. | 
| Oxidized glutathione in blood | Concentration of oxidized glutathione will be measured in red blood cells | At baseline. | 
| Protein carbonyls in serum | Concentration of protein carbonyls will be measured in serum. | At baseline. | 
| Total antioxidant capacity | Total antioxidant capacity will be measured in serum | At baseline. | 
| Malondialdehyde in serum | Concentration of malondialdehyde will be measured in serum. | At baseline. | 
| White blood cell count in blood | White blood cell count will be measured in blood. | At baseline. | 
| Insulin sensitivity | Insulin sensitivity will be assessed through an oral glucose tolerance test (OGTT) which involve ingesting glucose solution (75 g) with 5ml arterialized venous blood samples drawn at baseline and every 15 min during the first hour and every 30 min during the second hour over a 2-hour period. | At baseline. | 
| Glucose concentration in blood | Glucose concentration will be measured in plasma during the clinical trial. | At baseline and at 30 min, 60 min, 90 min, 120 min, 150 min and 180 min following protein ingestion. | 
| Insulin concentration in blood | Insulin concentration will be measured in plasma during the clinical trial. | At baseline and at 30 min, 60 min, 90 min, 120 min, 150 min and 180 min following protein ingestion. | 
| Body composition | Body composition will be measured using a dual-energy x-ray absorptiometry scanner (DEXA). | At baseline | 
| Body Mass Index (BMI) | Calculated as body mass (kg) divided by the height (m) squared. | At baseline | 
| Skeletal muscle index | Calculated as an appendicular lean mass (kg) divided by height (m) squared. | At baseline | 
| Grip strength | Using handgrip dynamometry (left and right arm) in a sitting position. | At baseline | 
| Functional performance | Functional performance will be assessed using the Short Physical Performance Battery (SPPB). | At baseline | 
| Lower limb muscle strength | Will be assessed by defining the 1 repetition maximum (1RM) on a knee-extension machine. | At baseline | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Low-grade systemic inflammation, Muscle protein synthesis, Proteasome activation, Anabolic signaling, Aging, Sarcopenia