2. Materials and Methods
dairy-based beverages, overweight, T2DM
OBESE, MINOR, RECRUITMENT, INSULIN RESISTANCE, BLIND, TYPE 2 DIABETES, INSULIN SENSITIVITY
Trial design: A randomized, double-blinded, placebo-controlled, parallel-group study design was employed, with the random allocation sequence to receive either HFC or LFC generated using an online calculator [This study was conducted according to the guidelines laid down in the Declaration of Helsinki 1973 (revised 1983) and all procedures involving human participants were approved by the University of Nottingham Medical School Ethics Committee. Written informed consent was obtained from all volunteers, and the protocol was registered at Participants: Pre-menopausal females who were overweight or grade 1 obese were recruited from the general Nottingham (UK) population through adverts placed in local newspapers. Those who fulfilled age (18–50 years), BMI (27–35 kg·mElevated HOMA-IR was a proxy to identify mild fasting IR and allow comparison with other studies, a value >1.5 having previously defined mild fasting insulin resistance in our laboratory. There was no upper limit for HOMA-IR, but a type 2 diabetes diagnosis based on fasting blood glucose concentration was set as an exclusion criterion. This cohort was identified as individuals at risk of developing T2DM, but who did not warrant pharmacological intervention. Eligibility criteria were not altered during the recruitment period.Intervention: Study visits took place during the first 10 days of participants’ menstrual cycle, in the morning after an overnight fast, and approximately 12 h after the final cocoa drink had been consumed. For those using a hormonal intrauterine device (3 participants), study visits were scheduled a month apart. Participants were weighed and a dual-energy X-ray absorptiometry (DEXA) total body composition scan was made (Lunar Prodigy, GE Medical, Hatfield, UK) to address the potential confounder of body composition on insulin sensitivity measures. The percentage of total tissue that was fat mass was calculated for two regions (using manufacturer-defined parameters): the femoral–gluteal region (‘Gynoid’ distribution), which contains predominantly subcutaneous fat, and the abdominal region (‘Android’ distribution), which is composed of subcutaneous and visceral fat. Participants then rested semi-supine and an intravenous cannula (Becton Dickinson, Helsingborg, Sweden) was inserted retrograde into a dorsal hand vein for arterialized-venous blood sampling, and antegrade into a forearm vein for 0.5 IU·mLA 3-h hyperinsulinemic (60 mIU·mParticipants returned for the post-intervention study visit approximately a month later (depending on the length of their menstrual cycle), having consumed the investigational product twice daily over this time, and the protocol described above was repeated. Over the intervention period, participants were instructed not to change their normal lifestyle and to maintain their usual physical activity levels.Investigational Product: The investigational products were dairy-based beverages made with either high-flavanol cocoa (609 mg cocoa flavanols, 95 mg (-)-epicatechin per serving; HFC group) or low-flavanol cocoa (13 mg of flavanols, 2 mg (-)-epicatechin per serving; LFC group). Products were supplied by Mars Incorporated (McLean, VA, USA) as powdered mixes, which were reconstituted by participants with 150 mL of warm water and consumed immediately following preparation. Powder sachets were identified by an anonymized code to blind volunteers and researchers to product allocation. The total amount of flavanols in each product was determined using the method described by Adamson et al. [Participants were given a 5-week supply of beverages and the supplementation period began on the day after the first laboratory visit. They maintained a daily record of product consumption over the intervention (to assess intake compliance), and weekly telephone contact was maintained throughout to identify any problems.Dietary Assessment: After recruitment, but before the first study visit, participants recorded all food intake, including snacks and drinks, in a 3-day (2 week and 1 weekend day) dietary record, using household measures to estimate portion size. Records were analyzed for daily macronutrient and energy intake using a food composition database (WISP V2, Tinuviel Software UK 2003), and an average over all 3 days of the recording period was taken. To reduce the impact of diet composition on the clamp-derived insulin sensitivity measures, the antecedent diet was then modified, by minor adjustments to the participant’s usual food choices, such that, in the 3 days prior to each study visit, 50% of the energy intake was provided by carbohydrates.In the third week of supplementation, participants completed a second 3-day dietary record to allow any change in diet, as a result of the intervention, to be characterized. Individuals were made aware of the extra dietary energy provided by the investigational beverages and advised to replace their current snack intake with the drink but were instructed to make no other changes to their habitual diet.Statistical Methods: Data were checked for normality of distribution and analyzed using SPSS version 25.0 (IBM, Armonk, NY, USA). Normally distributed data are described in the text as the mean with standard deviation in parentheses. Data that did not show a normal distribution are reported as the median with the 25th and 75th percentile in square brackets. Group characteristics pre-intervention were compared using unpaired Student’s t-test or non-parametric equivalent (Mann–Whitney U), and changes in variables at two time points but within groups were investigated using paired Student’s t-test or Wilcoxon signed ranks as appropriate. Cohen’s d was used to calculate standardized effect sizes [Blinding of all parties to the randomization group was maintained until after the study had been completed and data had been statistically analyzed and interpreted by the research team.Analytical Methods: Whole blood glucose was measured using the glucose oxidase enzymic method (Yellowsprings Inc., Yellow Springs, OH, USA; inter-assay and intra-assay coefficient of variation (CV) being 1%). Serum insulin was assessed using a human-specific radioimmunoassay (Merck Millipore, Billerica, MA, USA), with an intra-assay coefficient of variation (CV) of 4.41%, and an inter-assay CV of 10.53%.Sample size: Statistical power was calculated using a primary end point of steady-state glucose disposal ‘M’, with expected M values in insulin-resistant individuals being between 16 and 30 µmol·kg