Effect of Obesity-derived Cytokines on Protein Turnover and Carbohydrate Metabolism in Human Skeletal Muscle
Condition: Obesity; Sarcopenia
MeSH terms: Sarcopenia; Obesity
Study type: Interventional

Summary: 
      Obesity in humans has been shown to result in the increased release of small
      inflammatory-inducing proteins, called cytokines, from the fat cells of the body. The
      investigators are interested in the effects of these cytokines on the mechanisms that control
      muscle mass and metabolism in the obese human. Previous research from work in cells and
      animals has shown the cytokines reduce the synthesis of muscle proteins and simultaneously
      enhance their rate of breakdown, resulting in a loss of muscle mass. Furthermore, research
      suggests that the same cytokines may inhibit carbohydrate oxidation, a pivotal step in muscle
      metabolism. However, despite these potential negative consequences for skeletal muscle
      function, the effect of low-level and persistent inflammation as seen in obese humans,
      remains largely unknown.

      In the current study, the investigators plan to measure the rates of synthesis and breakdown
      of muscle proteins in conjunction with rates of carbohydrate oxidation in obese older
      participants, and compare them to rates determined in healthy non-obese individuals.
      Furthermore, participants will undergo a 12-week course of either pioglitazone, an insulin
      sensitiser often prescribed to type II diabetics, or a placebo. Pioglitazone has been shown
      previously to normalise the levels of cytokines in the blood of chronically inflamed
      individuals. By repeating after the 12-week intervention period the initial measurements
      described above, and by accurately determining the levels of the cytokines, the
      identification of the negative effects of obesity-induced inflammation in older adults on
      muscle metabolism will be determined.
    

Detailed description: 
      Recruitment of subjects:

      24 obese (but otherwise healthy) participants with elevated cytokine levels and 24 non-obese
      subjects with normal cytokine levels will be recruited. Following the first experimental
      visit (see below), participants will be divided in a double-blind fashion into four groups
      (n=12). In previous studies utilising similar low-grade inflammatory states where
      thiazolidinediones have been administered, significant differences (P<0.05) in processes
      under investigation have been observed with group sizes of 10. Therefore group sizes of 12
      have been selected to accommodate for the anticipated 15% dropout seen in long-term studies.
      Participants will be male, age matched (>55 years), non-smoking, live a sedentary lifestyle
      and non-vegetarian. Before taking part in the study, all subjects will undergo medical
      screening and complete a general health questionnaire. This will include measures of blood
      pressure, heart rate, 12-lead electrocardiogram (ECG), blood biochemistry and a blood
      clotting profile. For non-control subjects, inclusion into the study will be determined by
      measures of waist circumference (>100 cm), and inflammatory status (non-obese subjects with
      C-reactive proteins (CRP) plasma levels <1.35 μg.ml-1 and tumor necrosis factor-alpha (TNFα)
      plasma levels <3.3 pg.ml-1 and obese subjects with a CRP >1.35 μg.ml-1 and TNFα >4.1
      pg.ml-1). Following an explanation of the study and associated procedures, written consent
      will be obtained and subjects made aware that they are free to withdraw at any point.
      Subjects will be recruited via press advertisements from the local population.

      Experimental trials:

      At the start of a 12-week long study, muscle protein synthesis and leg protein breakdown will
      be determined in all subjects via the administration of stable-isotopes of leucine and
      phenylalanine concomitant to a two-stage insulin clamp to simulate going from the fasted to
      fed state. This will establish the baseline values for these measures prior to the 12-week
      intervention period. These measures will be achieved in a single visit with the execution of
      an established protocol for the determination of muscle protein synthesis and leg protein
      breakdown. In summary, to achieve this the following will be performed: a basal muscle biopsy
      from the thigh will be obtained after which serum insulin concentrations will be held at
      fasting levels and a further muscle biopsy obtained; once taken serum insulin concentrations
      will be elevated and mixed amino acids administered to simulate feeding followed by the
      taking of a third and final muscle biopsy. The taking of biopsies will allow for the
      determination of isotope incorporation and thereby the determination of muscle protein
      synthesis and leg protein breakdown rates.

      Following the first experimental visit, subjects will start a 12 week treatment period of
      either pioglitazone (30 mg•day-1) or a placebo, administered in a double-blind fashion.
      Subjects will be monitored at intervals throughout the period to ensure compliance and report
      unwanted side effects. After 12 weeks of drug or placebo administration, subjects will return
      to the laboratory for a repeat of the measurements determined in the first experimental visit
      (muscle protein synthesis and leg protein breakdown in response to simulated feeding).
      Circulating levels of select cytokines will be additionally examined in blood samples
      collected from subjects at the start of the two experimental visits (pre- and post- 12-week
      drug intervention period), thus allowing an assessment of any associated changes between
      systemic cytokine concentrations and muscle protein synthesis and leg protein breakdown.

      Study protocol:

      The study involves two visits separated by a 12-week drug intervention period. At each of the
      two visits muscle protein synthesis and leg protein breakdown rates in response to simulated
      feeding are to be assessed using an established experimental protocol described below:

      Subjects will be asked to abstain from alcohol and exercise for 48 h prior to each
      experimental visit and to arrive on the morning of the experimental visit in a fasted state.
      Subjects will be asked to rest in a semi-supine position while a cannula is inserted
      retrograde into a superficial vein on the dorsal surface of the non-dominant hand for blood
      sampling. The hand will be kept in a hand-warming unit (air temperature 50-55°C) to
      arterialise the venous drainage of the hand. A cannula will be placed in an antecubital vein
      in each forearm for the infusion of insulin, octreotide, glucose, amino acids and
      stable-isotope infusions of leucine and phenylalanine to simulate feeding in precise manner;
      a catheter placed in the femoral vein will allow, in conjunction with arterialised-venous
      (A-V) blood samples, arterial and venous blood sampling across the leg. To assess muscle
      protein synthesis and leg protein breakdown rates in response to feeding, serum insulin will
      initially be maintained at fasting concentrations by the administration of insulin (0.6
      mU•m-2•min-1) for 120 minutes and, to prevent endogenous insulin production, Octreotide (30
      ng•kg-1•min-1). Then, to simulate feeding, serum insulin concentrations will be increased
      equivalent to the fed state for 120 minutes (~40 mU•l-1) and 20g of mixed amino acids
      (Glamin® Fresenius-Kabi, UK) administered by infusion in conjunction with glucose to
      stabilise blood glucose concentrations (~4.5 mmol.l-1). Infusions of stable isotope versions
      of phenylalanine and leucine will be maintained throughout the experimental visit. Initially,
      a single priming dose of [2H5]phenylalanine (0.33 mg•kg-1 body mass, 98 atom % excess) and
      [1-13C]leucine (0.8 mg•kg-1 body mass, 99 atom % excess) will be administered, followed by a
      constant infusion of 0.5 mg•kg-1•h-1 phenylalanine and 1.0 mg•kg-1•h-1 leucine. The use of
      these two stable-isotopes in combination with muscle biopsies obtained from the vastus
      lateralis muscle of the leg at t = 0, 120 and 240 min and blood sampling throughout the
      visit, will allow muscle protein synthesis and leg protein breakdown rates in response to
      feeding to be calculated. At regular intervals throughout the experimental period,
      respiratory gas exchange will be monitored by measuring oxygen (O2) uptake and carbon dioxide
      (CO2) production using a ventilated hood indirect calorimeter (NutrEn, UK) to calculate the
      respiratory exchange ratio.

      After completion of the initial visit where baseline measures of muscle protein synthesis and
      leg protein breakdown in response to feeding have been established, the 12-week drug
      intervention period will begin. On completion of the 12-week intervention, the study visit
      described above will be repeated to determine the effects of the intervention on the outcome
      measures listed.

      Sample collection:

      At the start of each experimental visit, 8 ml of blood will be collected from a venous
      cannula into ethylenediaminetetraacetic acid (EDTA) vacutainers. Following centrifugation at
      4°C, the extracted plasma will be stored at -80°C to allow the later determination of
      circulating levels of select cytokines. During the insulin clamp, 1 ml of A-V blood will be
      obtained every 5 min for the monitoring of blood glucose concentration (YSI 2300 STATplus,
      Yellow Springs Instruments, USA). A-V blood (5 ml) will be collected at baseline and at +30,
      +75, +90, +105, +120, +150, +195, +210, +225, +240 min, allowed to clot, centrifuged, and the
      serum stored in liquid nitrogen. Insulin will be measured in these samples at a later date
      with a radioimmunoassay kit (Coat-a-Count Insulin, USA). Femoral venous and A-V blood samples
      (2 ml) will be taken for blood tracer measurements at -5, +75, +90, +105, +120, +195, +210,
      +225, +240 min, centrifuged at 4°C and stored at -80°C. Muscle biopsies will be obtained from
      the vastus lateralis muscle at 0, +120 and +240 min using the percutaneous needle biopsy
      technique. Muscle samples will be snap frozen in liquid nitrogen prior to the determination
      of [1-13C]leucine incorporation.

      After-care of the subjects:

      Auditing of the Investigator's past studies involving muscle biopsies and cannulation
      procedures has shown the procedures to be well tolerated by subjects and to have few
      significant complications. Scarring of the skin is minimal and invisible after a few months.
      All muscle biopsies are obtained under sterile conditions by medically qualified staff and
      after the injection of local anaesthetic. All subjects are carefully monitored before,
      during, and after the procedure to ensure that they are comfortable at all times. After each
      biopsy, the skin is closed with a sterile adhesive strip and covered with a waterproof
      adhesive dressing. On removal of the cannulae, pressure is applied to stop bleeding, and the
      sites dressed with a waterproof adhesive dressing. Subjects are given a leaflet before they
      leave the laboratory containing after-care information and contact details of our clinicians
      should they need advice. Further, subjects will be contacted by telephone on weeks 2, 6 & 10
      to confirm the absence of any adverse effects of the drug administration. Subjects will also
      be provided with contact details that they can use should they have any concerns.

      Measurements and analysis:

      Muscle biopsy samples and blood samples shall be measured for tracer incorporation, allowing
      rates of muscle protein synthesis and breakdown to be calculated. Collected blood samples
      will be analysed for circulating levels of cytokines. By performing these measurements pre-
      and post- pioglitazone treatment, the consequences of inflammation in aged individuals on
      muscle metabolism will be determined. Statistical analysis shall be performed using analysis
      of variance (ANOVA).
    

Intervention name(s): Pioglitazone; Insulin; Octreotide; Glucose; Mixed Amino Acids; [2H5]phenylalanine; [1-13C]leucine
Intervention type(s): Drug; Drug; Drug; Drug; Drug; Other; Other
Intervention description(s): Described elsewhere; Administered to simulate the feeding response in study visits. Description of amount administered provided elsewhere.; Administered to simulate the feeding response in study visits. Description of amount administered provided elsewhere.; Administered to simulate the feeding response in study visits. Description of amount administered provided elsewhere.; Administered to simulate the feeding response in study visits. Description of amount administered provided elsewhere.; Administered to allow the determination of leg protein breakdown rates in study visits. Description of amount administered provided elsewhere.; Administered to allow the determination of muscle protein synthesis rates in study visits. Description of amount administered provided elsewhere.
Other intervention name(s): Actos; Actrapid; Sandostatin; Dextrose; Glamin
Intervention MeSH terms: Pioglitazone; Octreotide
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Triple (Participant, Investigator, Outcomes Assessor)
Primary purpose: Basic Science