Effects of Acute Energy Deficit and Aerobic Exercise on Muscle Quality
Condition: Energy Supply; Deficiency; Energy Balance; Energy Availability
Study type: Interventional

Summary: 
      10 healthy, male, participants will complete a a 5-day baseline assessment (days -5 to -1)
      and two consecutive 5-day periods of controlled exercise to increase oxidative capacity (3
      days of aerobic exercise per period, 15 kcal/kg FFM/day energy expenditure cycling) and
      energy intake (15 days in total, with a testing session on morning 16). This will achieve
      states of energy balance (EB; energy availability - EA - 45 kcal/kg of fat free mass
      (FFM)/day), required for weight maintenance (days 1 - 5), followed by energy deficit (ED; EA
      10 kcal/kg FFM/day), required for weight loss on days 6 - 10.

      Over the data-collection period, participants will consume deuterium (D2O) tracer to
      facilitate dynamic proteomic profiling to assess the impact of the intervention on muscle
      quality (primary outcome measure). Muscle biopsies will therefore be collected on days -5, 1,
      6 & 11, alongside daily saliva samples, and venous blood collection on days -5, 1, 3, 5, 6,
      8, 10 & 11. These samples will be used to assess further, secondary, outcome measures
      including alterations in intra-muscular lipid profiles (lipid droplet content, morphology and
      lipid-droplet associated proteins in different subcellular compartments [intermyofibrillar vs
      subsarcolemmal]), alterations in blood metabolites and hormones and skeletal muscle glycogen
      concentrations. Changes in body mass, body composition and RMR will also be assessed.
    

Detailed description: 
      Justification for the research:

      Weight-loss strategies that use energy restriction alone can lead to impaired muscle mass,
      which can also further impair health of individuals with metabolic conditions, such as type 2
      diabetics. Skeletal muscle mass and function are key to maintaining a healthy metabolism and
      quality of life throughout the lifespan. The combination of exercise and calorie restriction
      is a powerful intervention for reducing body weight and improving the metabolism and health
      status of healthy, overweight and obese individuals. The investigators have previously shown
      that skeletal muscle protein synthesis is reduced with energy restriction and that
      resistance-type exercise can reverse this negative effect. Aerobic-type exercise also has the
      capacity to stimulate muscle protein synthesis and improve the quality of muscle by
      increasing mitochondrial protein synthesis. Nonetheless, how energy deficit overlayed on top
      of aerobic exercise modulates skeletal muscle quality is not well characterised. Furthermore,
      existing studies have looked only into mixed (unspecific) protein synthesis of different
      intracellular compartments without providing details on how protein synthesis for specific
      proteins is regulated.

      This project will provide novel data to unravel the mechanisms behind the positive effect of
      energy restriction and concomitant aerobic exercise on skeletal muscle quality.

      Aerobic exercise alone is a well-established intervention to increase mitochondrial capacity
      and skeletal muscle function, but the effect on skeletal muscle of overlaying energy
      restriction while performing aerobic exercise is not well characterised. Recent findings in
      Rhesus monkeys, whose physiology responds in a very similar way to that of humans, has shown
      that life-long caloric restriction has a profound positive effect on skeletal muscle. These
      findings show that caloric restriction not only maintains contractile content of muscle, but
      also rescues the age-related decline of skeletal muscle mitochondrial content and capacity.
      However, physical activity in this study was not controlled and appeared to be higher in the
      caloric restriction group, representing an important confounding factor. Research in humans
      addressing similar questions so far has been less clear. Pronounced weight loss through
      energy restriction alone (10% total body weight in ~7.5 wk) in obese women lead to a decrease
      in muscle mitochondrial content. In stark contrast, using a milder restriction of 25% of
      total energy alone or combined with exercise during 6 months in overweight individuals showed
      increased expression of genes encoding mitochondrial proteins in both groups. However,
      despite these promising findings suggesting that weight-loss combined with exercise will
      enhance skeletal muscle metabolism, there are currently no strong data to provide support on
      the use of concomitant energy restriction and aerobic exercise with in-depth analysis of its
      physiological and molecular effects in humans.

      The proposed study herein will include a short period of tightly controlled exercise and
      dietary intake to show that energy deficit while performing aerobic exercise results in
      further benefits to muscle metabolic adaptation. The current project builds on from the
      investigator's previous and recent research findings to directly address this question. This
      research has shown that resistance exercise during 30% daily energy deficit rescues the
      decrease in mixed (unspecific) myofibrillar protein synthesis observed with energy deficit
      alone. Moreover, the investigator's research has shown that aerobic exercise up-regulates
      specific proteins in skeletal muscle mitochondria in rodents; that skeletal muscle lipid
      droplet profile is responsive to exercise and nutrition; that nutrition modulates the
      cellular response to aerobic exercise and, importantly; that aerobic exercise after
      short-term (~14 hours) energy deficit can up-regulate markers of mitochondrial biogenesis in
      skeletal muscle and improve metabolic control in humans.

      Objectives & Hypothesis:

      The primary research question associated with this study is: 'What is the combined effect of
      an energy deficit and aerobic exercise training on muscle quality (synthesis rates of
      individual, sarcoplasmic and mitochondrial proteins) and intramuscular lipid dynamics over a
      five-day period in healthy males?'

      Further objectives:

        1. Identify the mechanisms by which a short-term energy deficit (achieved through
           concomitant energy restriction and exercise) can regulate individual muscle protein
           turnover (synthesis, breakdown and abundance), intramuscular lipid dynamics, metabolites
           and muscle glycogen content during weight-loss.

        2. Provide novel evidence for the capacity of a potato-based diet as an effective source of
           nutrients to promote positive skeletal muscle and metabolic adaptations during weight
           loss.

      Study hypothesis:

      Short-term energy restriction with concomitant aerobic exercise will increase the quality and
      quantity of skeletal muscle proteins related to mitochondrial capacity, improve skeletal
      muscle intracellular lipid droplet profile and modulate blood-borne markers of metabolic
      health compared to exercise without energy deficit.

      Study Methods:

      Participants will first undertake testing for characterisation (detailed below) followed by a
      5-day baseline assessment (days -5 to -1) and two consecutive 5-day periods of controlled
      exercise to increase oxidative capacity (3 days of aerobic exercise per period, 15 kcal/kg
      FFM/day energy expenditure running) and energy intake (15 days in total, with a testing
      session on morning 16). This will achieve states of energy balance (EB; energy availability -
      EA - 45 kcal/kg of fat free mass (FFM)/day), required for weight maintenance (days 1 - 5),
      followed by energy deficit (ED; EA 10 kcal/kg FFM/day), required for weight loss on days 6 -
      10. This experimental design, as well as energy intake and expenditure, replicates what the
      investigators have previously used to successfully determine other physiological effects of
      ED on skeletal muscle. Five days of EB and EA will allow for tight control of dietary intake
      and will be sufficient to detect changes in the main parameters being investigated.

      Participant characterisation testing will be ~15 days prior to the start of baseline
      assessment to determine compliance with inclusion criteria, fitness levels (maximal oxygen
      consumption and lactate threshold [via finger-prick capillary samples]) and body composition.
      At baseline assessment a resting muscle a biopsy will be taken from the vastus lateralis
      (quadriceps), prior to ingestion of the tracer deuterium oxide (D2O) through the following 15
      days to allow dynamic proteomic profiling of skeletal muscle. A total of 4 biopsies will be
      taken per participant, which is in line with the investigator's prior research. The baseline
      assessment is used for enrichment of D2O for dynamic proteomic profiling assay and ensuring
      regular physical activity of individuals matches requirements of the study. Throughout each
      5-day experimental period, muscle biopsies (days -5, 1, 6 & 11), daily saliva samples
      (non-stimulated, collected at home) and regular venous blood samples (days -5, 1, 3, 5, 6, 8,
      10 & 11) will be collected for the assessment of dynamic proteomic profiling, fibre-type
      specific lipid droplet profiling, skeletal muscle glycogen and D2O, and blood-borne hormones
      and metabolites related to weight loss. Resting Metabolic Rate (RMR; assessed via indirect
      calorimetry) and body composition assessment will be conducted using dual X-ray
      absorptiometry (DXA) scans on days -5, 1, 6 & 11 of the intervention. Bioelectrical impedance
      analysis (BIA) will also be used to assess body composition at each lab visit (days -5, 1, 3,
      5, 6, 8, 10 & 11).

      Dietary interventions and energy balance:

      The diet will follow the reference daily intake of nutrients to provide ~60, 20 and 20% of
      energy from carbohydrates, fat and protein, respectively. In line with funding requirements,
      the percentage of total energy from potato-based sources will be >60% during EB and >65%
      during ED. Potatoes will be cooked in a range of different ways that do not add significant
      amount of energy such as boiled, microwaved, baked, etc.

      Energy availability manipulation:

      Energy availability (EA), which is defined as energy intake minus energy expenditure from
      exercise - normalised to fat free mass - is used as a key parameter to determine energy
      balance. The investigators are the first to have shown that values under 30 kcal/kg/FFM can
      modulate skeletal muscle responses, which is in line with other research on a range of
      metabolic responses to EA. Based on this recent research, which used 20 kcal/kg FFM/day for
      ~14 hs, the current intervention will induce a more pronounced energy deficit for longer. The
      average energy intake across the 5 days for an average 85 kg (70 kg FFM) participant will be
      3780 kcal/day for EB and 1330 kcal/day ED. This will achieve an approximate cumulative energy
      deficit of 12250 kcal over the 5-day period resulting in approximately ~1.5 kg of body mass
      loss from net tissue, given that ~7450 kcal are necessary to lose 1 kg of net tissue. Water
      loss will account for an additional ~1 kg of the loss in body weight which will be a
      consequence of a decrease in endogenous carbohydrate (glycogen) stores, to which water binds,
      as well water fluctuation due to sodium changes. Skeletal muscle glycogen is also a regulator
      of the muscle qualitative aspects and will also be investigated.

      Subjects. A normal population will allow the researchers to determine the physiological
      responses to this intervention. Ten young (18-40 years) healthy, regularly exercising men
      with a body fat percentage ~18-26%.

      Quality:

      The researcher's associated with this study and associated with the review of the study
      protocol are all members of staff (or a PhD Student) within the Liverpool John Moores
      University Research Institute for Sport & Exercise Sciences (RISES). In the 2014 RISES (LJMU)
      submitted 34.75 FTE (full time equivalent) to Unit of Assessment 26 (UoA26) and attained a
      GPA (Grade Point Average) of 3.57. Placed second on GPA in the UoA, RISES became the leading
      centre for Sport and Exercise Science Research Quality in the UK (4* - 61% of all activity
      world leading, 3* - 36% of all activity internationally excellent standard). RISES submitted
      the largest volume of 4* outputs (n=60) in the UoA, had 90% of the impact activity rated at
      4* and had 100% of the environment rated as 4*. Importantly, out of 1,911 submissions in all
      36 UoA's RISES came 11th in the entire UK for GPA achieved at REF2014 putting RISES (LJMU)
      amongst Oxford, UCL, LSE and Cambridge in the league tables for this metric.
    

Intervention name(s): Energy Balance; Energy Deficit; Baseline Assessment
Intervention type(s): Dietary Supplement; Dietary Supplement; Dietary Supplement
Intervention description(s): Energy balance phase to elicit weight-maintenance; Energy deficit phase to elicit weight-loss; Free-living assessment of energy status
Allocation: Non-Randomized
Intervention model: Sequential Assignment
Intervention model description: Participants will complete a 5-day free-living baseline assessment period (days -5 to -1) and two consecutive 5-day periods (days 1 - 10, with a final assessment on the morning of day 11) of controlled exercise to increase oxidative capacity (3 days of aerobic exercise per period, 15 kcal/kg FFM/day energy expenditure cycling) and energy intake.
This will achieve states of energy balance (EB; energy availability - EA - 45 kcal/kg of fat free mass (FFM)/day), required for weight maintenance (days 1 - 5), followed by energy deficit (ED; EA 10 kcal/kg FFM/day), required for weight loss on days 6 - 10.
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: Dynamic proteomic profile; Skeletal muscle; Intra-muscular lipid profiles; Skeletal muscle glycogen; Glucose; Insulin; Leptin; Ghrelin; Testosterone; Adiponectin