Effects of Chronic Dietary Exposure to Branched Chain Amino Acids
Condition: Type 2 Diabetes Mellitus With Features of Insulin Resistance
MeSH terms: Diabetes Mellitus, Type 2; Insulin Resistance
Study type: Interventional

Summary: 
      Branched-chain amino acids (BCAA) belong among nutrients strongly linked with insulin
      sensitivity (IS) measures. Their exact effect on IS appears to be dependent on various
      conditions that remain to be better defined . The aim of the current study was to investigate
      effects of chronic increase of BCAA intake on IS in two groups of healthy subjects differing
      in their basal consumption of BCAA, i.e. vegans and omnivores. Interventional trial was
      designed where vegans and omnivores were separately interveined with 15 (women) or 20 (men)
      grams of BCAA daily. Examinations of participants are intended to be done at baseline, after
      the intervention and wash-out period. Examinations (anthropometry,
      hyperinsulinaemic-euglycaemic clamp, arginine test) and blood analyses will be performed at
      baseline, after the intervention and after a 6 month wash-out period. Samples of subcutaneous
      abdominal adipose tissue (AT) and skeletal muscle (SM, vastus lateralis) obtained before and
      after the intervention will be used for various analyses (mRNA levels of selected metabolic
      markers, fatty acid composition, mitochondrial activity).
    

Detailed description: 
      Subjects and methods Listed elsewhere Intervention Listed elsewhere

      Clinical examination Each subject underwent a basic medical check-up with anthropometric
      examination (height (m), weight (kg), BMI (kg/m2), waist circumference (cm), waist-hip
      ratio). Body composition was measured using bioimpedance analysis (BIA, Nutriguard-M, Data
      Input GmbH, Germany). Each of above mentioned measurements was performed three times and mean
      was recorded.

      Dietary assessment Each participant filled in a prospective questionnaire, where dietary data
      from 3 days were collected (2 working days, 1 weekend day). For dietary intake calculations
      Nutridan program was used. As nutritional data of some special vegan products are not
      available in the database, vegans were asked to collect packages from these products with
      producer declared nutritional content, and these data were used for calculations.
      Saccharides, lipid and protein intake were calculated separately. BCAA content of dietary
      proteins was estimated using nutritional database.

      Physical activity assessment Physical activity was assessed in the whole sample using Baecke
      questionnaire for habitual physical activity [1], that correlates well with maximum oxygen
      consumption (VO2max)[2]. Maximal exercise test was performed in each subject on an
      electromagnetically braked bicycle ergometer (Ergoline 800, Bitz, Germany) to determine peak
      oxygen uptake (Vo2peak). An initial workload of 50W was increased by 25 W every minute
      continuously until fatigue despite the verbal encouragement. Oxygen uptake was measured using
      Vmax, Sensor Medics (Yorba Linda, CA). Heart rate was monitored continuously.

      Laboratory analysis Peripheral venous blood was drawn from each subject after 12 hours of
      fasting. Parameters of glucose homeostasis were assessed in certified University hospital
      laboratory: plasma glucose using hexokinase reaction (kit KONELAB, Germany), HbA1c using
      high-pressure liquid boronate afinit chromatography (Primus corporation), insulin using solid
      phase competitive chemiluminescent enzyme immunoassay (Immulite 2000). Lipid profile: total
      cholesterol and triglycerides were measured using enzymatic method (kit KONELAB, Germany),
      HDL-cholesterol using PEG modified enzymatic measurement (kit ROCHE, Switzerland). Plasma
      levels of free fatty acids (FFA) were measured using method already described [12]. Briefly,
      FFA were extracted together with neutral lipids using isooctan and cleaned by reverse
      extraction. FA obtained this way were derivatized to methylesters and subsequently analyzed
      using gas chromatography (GC). Serum AA levels were determined using capillary
      electrophoresis (CE) with contactless conductivity detection, which has been already
      described in details [3, 4]. CE measurements were carried out using HP3DCE system (Agilent
      Technologies, Waldbronn, Germany) equipped with a built-in contactless conductivity detector.

      Insulin sensitivity and secretion IS was assessed using 2-hours hyperinsulinaemic euglycaemic
      clamp, method described elsewhere[5]. Clamp was performed after 12-hours fasting in a
      standard insulin dose 1mIU/kg/min and an infusion of 15 % glucose solution was used. Mean
      infusion rate in steady-state of the clamp (6 consecutive measurements) was used for
      measurements. Glucose disposal was expressed as metabolic clearance rate (MCR, ml.kg-1.min-1)
      after correction for changes in glucose pool in extracellular fluid (space correction) and
      insulin sensitivity index (MCR divided by stady state insulinaemia, MCR/I,
      ml.kg-1.min-1/mU.l-1). Insulin secretion was assessed by IV arginine test as already
      described [6] performed on a different day (at about 7 days in between) from glucose clamp.
      Acute insulin response was calculated as the incremental trapezoidal area for insulin during
      the 30 min of the test.

      Muscle biopsy, respiratory chain, and citrate synthase enzymes activities Skeletal muscle
      sample from vastus lateralis muscle was performed in every participant using a standard
      Bergström technique[7, 8]. Biopsy was performed at fasting conditions. About 200 mg of wet
      weight was obtained. The sample was immediately microdissected, weighed, divided for
      respective analyses and snap-frozen in liquid nitrogen and stored in -80oC until analyses.

      Mitochondrial respiratory chain (RC) enzymatic activity and citrate synthase (CS) activity
      were measured spectrophotometrically in muscle homogenates. Muscle homogenates were prepared
      as described [9]. Briefly, about 50 mg of SM was cut into small fragments and homogenized
      (glass-glass grinder) in 20 volumes of ice-cold homogenization buffer (250 mM sucrose, 20 mM
      Tris, 40 mM KCl, 2 mM EGTA, protease inhibitor cocktail, pH 7.4). The homogenates were then
      centrifuged for 1 min at 600g at 4°C and the supernatants were immediately used for analysis.
      Protein concentration was measured in aliquots using the bicinchoninic acid (BCA) assay
      (Sigma). Activity of complex I-IV and CS was determined accordingly to previously published
      protocols [10-11], that were modified for measurement in a microplate reader (Infinite M200
      PRO, Tecan). All measurements were made in tetraplicates. Activities of RC and CS were
      expressed as nmol/min/mg of total proteins, except for complex IV, which was expressed as Δ
      log (A550)/min/mg of total proteins. The enzymatic activities of the RC complexes were also
      normalized to the activity of CS, which is used as a marker of the abundance of mitochondria
      within a tissue.

      Adipose tissue biopsy Subcutaneous abdominal adipose tissue (SAAT) biopsy was performed in
      every participant. Bergström needle was used to obtain samples of subcutaneous fat from
      paraumbilical area as already described [12]. Biopsy was performed at fasting conditions.
      About 300 mg of wet weight was obtained. The sample was immediately microdissected, weighed,
      divided for respective analyses and snap-frozen in liquid nitrogen and stored in -80oC until
      analyses.

      Quantitative real time PCR (RT-qPCR) Total RNA from tissues was isolated using Lipid Tissue
      and Fibrous Tissue RNeasy Mini Kits (Qiagen). RNA concentration was measured by Nanodrop1000
      (Thermo Fisher Scientific, Wilmington, USA). RNA was treated with DNAse I (Invitrogen,
      Carlsbad CA, USA) to remove any contaminating genomic DNA. cDNA was prepared from 200- 600 ng
      of RNA using High Capacity cDNA archive kit (Applied Biosystems, Carlsbad CA, USA).
      Equivalent of 5 ng of RNA was used for Real Time PCR reactions using Fast Advanced master mix
      and Gene expression assay (IRS1, GLUT4, BCKDHA, BCKDHB, ACOX, CPT1b, PLIN1, PLIN2, PLIN5,
      FASN, SCD1, DGAT2, PPAR-γ; Applied Biosystems). All samples were run in duplicates. Gene
      expression of target genes was normalized to expression of RPS13 (glucuronidase, beta) and
      fold change of expression was calculated using delta delta Ct method.

      Fatty acid spectrum in AT Fatty acid spectrum in AT was assessed using gas chromatography, a
      method already described by Lepage and Roy [13] with modifications by Rodriguez-Palmero et
      al. [14] In brief, the method involves a chloroform/methanol extraction of freeze-dried AT to
      isolate lipids and subsequent transesterification or esterification of FA bound to lipids to
      form methyl esters, which were then analyzed using gas chromatography.

      Statistical analysis Data are presented in text, tables, and figures as means ± SD with 95 %
      CI and p-values <0.05 were considered statistically significant. Student t-test was used in
      observational samples for normally distributed data. Wilcoxon matched pairs test was used
      when data were not normally distributed. Intervention samples were compared using a general
      linear model to test the statistical significance of differences between groups. A mixed
      model ANOVA was used to assess group × time interaction. Within each group time effects of
      the intervention were assessed using repeated measures ANOVA with Bonferroni's multiple
      comparison. Where there were only two data sets within each group (i.e. only baseline and
      intervention data as in data obtained from tissue biopsies) paired t-tests were used for
      normally distributed data and Wilcoxon test when data were not normally distributed.
      Pearson's correlation coefficient was calculated to express relationship between changes from
      baseline to intervention for normally distributed data and Spearman's correlation coefficient
      was calculated when data were not normally distributed. Statistica 9.0, StatSoft, Inc. USA
      was used to perform all statistical procedures.
    

Intervention name(s): branched chain amino acids
Intervention type(s): Dietary Supplement
Intervention description(s): 15 (women) or 20 (men) grams of BCAA daily for 3 months
Allocation: Non-Randomized
Intervention model: Crossover Assignment
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: insulin resistance; bcaa; mitochondrial biogenesis