Effect of Meal Frequency on Insulin Resistance in Subjects With Type 2 Diabetes
Condition: Diabetes Mellitus, Type 2
MeSH terms: Diabetes Mellitus; Diabetes Mellitus, Type 2; Insulin Resistance
Study type: Interventional

Summary: 
      Aims and priorities of the project The purpose of this study is to

        1. test the effect of frequency of meals (six vs. two meals daily with the same daily
           caloric restriction of -500 kcal/day) on insulin sensitivity, insulin secretion, and
           hepatic fat content.

        2. characterize some of the mechanisms of action of different frequencies of meals (amount
           of visceral fat, hepatic fat content, serum concentrations of adipokines, gut hormones,
           oxidation stress markers).

        3. test the ability of the participants to maintain hypocaloric diet on both regimens when
           educated and left to prepare their meals alone in comparison with those for whom all
           meals during the study will be provided.

      It will be a randomized, crossover study, where 50 individuals with type 2 diabetes will
      change in a random order two regimens: six, and two meals a day. Each testing period will
      take three months.

      Glucose and lipid metabolism and its regulation will be thoroughly tested at start, and after
      each 3-months-period (meal test, hyperinsulinemic isoglycemic clamp, indirect calorimetry,
      MRI scan of the liver, DXA scan, serum concentration determination of selected adipokines,
      gut hormones, and oxidation stress markers).

      Hypothesis The investigators hypothesize that low plasma insulin levels (as achieved by
      periods of fasting) will reduce insulin resistance and hepatic lipid content. In contrast,
      frequent meals (and consequent higher plasma levels of insulin) will predispose to
      non-alcoholic fatty liver disease and insulin resistance. The investigators further
      hypothesize that the participants will increase their caloric intake with increased meal
      frequency (in spite of thorough education) when left to prepare their meals in comparison
      with those for whom all meals will be provided.
    

Detailed description: 
      Aims and priorities of the project The purpose of this study is to

        1. test the effect of frequency of meals (six vs. two meals daily with the same daily
           caloric restriction of -500 kcal/day) on insulin sensitivity, insulin secretion, and
           hepatic fat content.

        2. characterize some of the mechanisms of action of different frequencies of meals (amount
           of visceral fat, hepatic fat content, serum concentrations of adipokines, gut hormones,
           oxidation stress markers).

        3. test the ability of the participants to maintain hypocaloric diet on both regimens when
           educated and left to prepare their meals alone in comparison with those for whom all
           meals during the study will be provided.

      It will be a randomized, crossover study, where 50 individuals with type 2 diabetes will
      change in a random order two regimens: six, and two meals a day. Each testing period will
      take three months.

      Glucose and lipid metabolism and its regulation will be thoroughly tested at start, and after
      each 3-months-period (meal test, hyperinsulinemic isoglycemic clamp, indirect calorimetry,
      MRI scan of the liver, DXA scan, serum concentration determination of selected adipokines,
      gut hormones, and oxidation stress markers).

      Hypothesis The investigators hypothesize that low plasma insulin levels (as achieved by
      periods of fasting) will reduce insulin resistance and hepatic lipid content. In contrast,
      frequent meals (and consequent higher plasma levels of insulin) will predispose to
      non-alcoholic fatty liver disease and insulin resistance. The investigators further
      hypothesize that the participants will increase their caloric intake with increased meal
      frequency (in spite of thorough education) when left to prepare their meals in comparison
      with those for whom all meals will be provided.

      Key words Insulin resistance, meal frequency, type 2 diabetes mellitus, non-alcoholic fatty
      liver disease, adipokines, gut hormones, oxidative stress Methodology Study design: the
      investigators will use the design of a randomized, crossover study, where 50 individuals with
      type 2 diabetes will change in a random order the frequency of their meals: six, and two
      meals a day. Caloric restriction will be the same (-500 kcal/day). Each testing period will
      take three months. For one half of the participants all meals during the study will be
      provided. The other half will be thoroughly educated how to maintain their daily caloric
      intake during both regimens and they will prepare their meals alone.

      Study group: 50 individuals with type 2 diabetes treated by diet only or oral hypoglycemic
      agents, diabetes duration at least 1 year, both men and women, age 30-65 years, BMI 27-50
      kg/m². The subjects will be explained aims, methods and risks of the study and they will sign
      informed consent (Appendix 1).

      Regimens: On the six-meals-per-day-regimen, participants will be asked to divide their total
      caloric intake into six meals and to eat every two or three hours. On the
      two-meals-per-day-regimen, they will divide their total caloric intake into two meals: the
      first meal will be eaten between 6 and 10 a.m., the second one between noon and 4 p.m.

      Physical activity: Participants will be asked not to change their exercise habits during the
      study. Physical activity will be monitored using pedometers and standardized questionnaires:
      International Physical Activity Questionnaire (IPAQ), and Baecke questionnaire.

      Procedures:

      At the beginning (week 0), and at the end of every 3 months (week 12, and 24), the following
      procedures and measurements will be performed at each subject (three times in each subject):

        1. Common anthropometric investigations (body weight, BMI, waist and hip circumference),
           blood samples will be taken for laboratory assessments (common laboratory tests,
           parameters of glucose and lipid metabolism, chosen adipokines, oxidative stress markers,
           gut hormones, fatty acid composition in serum phospholipids etc.- see analytic methods).

        2. Meal test for glucose tolerance assessment after standard breakfast /baguette Crocodille
           Cheese Gourmet - 180g, energy 452,8 Kcal/1895,7 kJ, composition: carbohydrates 49,2 g
           (44,55%), proteins 18,5 g (16,74%), lipids 18,8 g (38,7%), of which saturated 6,8 g,
           monounsaturated 6,0 g, polyunsaturated 5,0 g/. Blood samples for the assessment of
           glycemia, C- peptide, immunoreactive insulin (IRI) will be taken in 0, 30, 60, 120 and
           180 minutes after breakfast.

        3. Hyperinsulinemic (1 mU/kg/min) isoglycemic clamp (HIC) 3 hours long with indirect
           calorimetry. This method allows exact quantification of insulin resistance and energy
           substrates utilisation.

        4. MRI (magnetic resonance imaging) scan of the liver to measure the hepatic fat content.

        5. DXA (Dual energy X-ray absorptiometry) scan to measure total body composition and fat
           content.

        6. Insulin secretion and glucose control will be measured during a one-day-stay in the
           hospital at the end of each regimen. Blood samples will be taken every three hours
           during the whole day. Urine will be collected during the whole day to measure
           microalbuminuria and C-peptide waste.

      Analytic methods:

      Plasma concentrations of selected adipokines (resistin, adiponectin total, HMW- adiponectin,
      TNFalfa and leptin) and other cytokines and proteins (FABP) will be measured enzymatically
      using standard kits (ELISA, Linco, USA). Plasma levels of gut hormones will be measured
      enzymatically usig standard kits (Milliplex, Millipore, USA). Parameters of lipid
      peroxidation will be determined according to the levels of TBARS by the reaction with
      thiobarbituric acid and according to the levels of conjugated dienes.

      The level of reduced and oxidised glutathione will be determined using HPLC method with
      fluorescence detection. Ascorbic acid will be determined spectrophotometric reaction with
      dinitrophenylhydrazine. Concentrations of a- and g-tocopherol in serum will be determined by
      reverse-phase high performance liquid chromatography (HPLC) with fluorescence detection
      according to the modified method of Catignani and Biery. The activity of SOD will be analyzed
      by the reaction of blocking nitrotethrazolium blue reduction and nitrophormasane formation.
      Catalase activity measurement is based on the ability of H2O2 to produce with ammonium
      molybdate the color complex detected spectrophotometrically. The activity of gluthathione
      peroxidase will be monitored by oxidation velocity of gluthathione by Ellman reagent. Serum
      glucose will be analysed using the glucose-oxidase method Beckman Analyzer (Beckman
      Instruments Inc., Fullerton, CA, USA), IRI will be determined by radioimmunoassay using an
      IMMUNOTECH Insulin IRMA kit (IMMUNOTECH as, Prague, Czech Republic), C-peptide using an
      IMMUNOTECH C-Peptide IRMA kit (IMMUNOTECH as, Prague, Czech Republic) and glycated hemoglobin
      will be measured by a Bio-Rad Haemoglobin A1c Column Test (Bio-Rad Laboratories GmbH, Munich,
      Germany).

      Fatty acid pattern in serum phospholipids will be measured after lipids extraction according
      to Folch and separation of lipid fractions by thin-layer chromatography. The methylesters of
      fatty acids will be separated by gas chromatography.

      Eventual effects of different meal frequency regimens will be evaluated due to chosen gene
      polymorphisms.

      Statistic analysis: Will be done with the use of ANOVA tests, pair and unpair t- tests and
      other statistic methods using standard statistic programs. Estimate of the number of subjects
      to be recruited was done using power analysis of repeated measurements via statistic software
      PASS 2005 (Number Cruncher Statistical Systems, Kaysville, UT, USA). Factors included in this
      model are interindividual factors (control vs. experimental group), intraindividual factors
      (individual time stadium in the study) and interaction between factors (divergence degree
      between time profiles in control and experimental group).

      Time frame: December 2010-February 2011 recruitment of the participants March 2011 start of
      the study Group A: 12 weeks 6 meals/day followed by additional 12 weeks of 2 meal/day Group
      B: 12 weeks 2 meals/day followed by additional 12 weeks of 6 meal/day September 2011 end of
      the study

      All measurements will be done at start, week 12 and week 24.
    

Intervention name(s): Meal frequency (6 meals vs. 2 meals/day); 6 meals/day followed by 2 meals/day
Intervention type(s): Behavioral; Behavioral
Intervention description(s): 6 meals/day for 12 weeks followed by 2 meals/day for 12 weeks at the same caloric restriction (-500 kcal/day); 2 meals/day for the first 12 weeks followed by 6 meals/day for additional 12 weeks at the same caloric restriction (-500 kcal/day)
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: meal frequency; type two diabetes