Effects of Conjugated Linoleic Acid and Metformin on Insulin Sensitivity in Obese Children

Randomized Clinical Trial

Nayely Garibay-Nieto; Gloria Queipo-García; Flor Alvarez; Mayra Bustos; Erendira Villanueva; Fernando Ramírez; Mireya León; Estibalitz Laresgoiti-Servitje; Ravindranath Duggirala; Teresa Macías; Sergio Cuevas; Abel Jalife; Miguel Fonseca-Sánchez; Fabiola Serratos; Juan Carlos López-Alvarenga


J Clin Endocrinol Metab. 2017;102(1):132-140. 

In This Article

Subjects and Methods

We performed a randomized, double-blinded, 16-week placebo (PLB)-controlled trial in the Pediatric Obesity Clinic at the Pediatrics Department of Hospital General de México (Mexico City, Mexico).

Patients with obesity aged 8 to 18 years who had not been previously intervened and had optimal psychological health were included in the study. Obesity was defined using Centers for Disease Control and Prevention criteria [body mass index (BMI) ≥ 95th percentile]. Exclusion criteria included BMI≥35 kg/m2, genetic or endocrine obesity, a systemic illness, diabetes or prediabetes (according to American Diabetes Association criteria),[15] and the use of weight loss medications that could modify lipids and glucose concentrations. The study (no. DI/11/311/04/108) was approved by the hospital's institutional review board; additionally, it was registered in ClinicalTrials.gov (no. NCT02063802).

All participants were included in the standardized healthy lifestyle program addressed to children and their parents. This 4-month program consisted of a monthly visit that included a 1-hour structured physical activity session (coordinated by a physical trainer), followed by a psychoeducational group session. The following information was presented to all participants: (a) description of a balanced and healthy nutrition, (b) emotion-related eating behavior and family support, (c) the benefits of physical activity, and (d) obesity-related comorbidities. These sessions were coordinated by nutritionists, psychologists, pediatricians, pediatric endocrinologists, and a physical trainer. Afterward, all patients held a medical consultation to evaluate their anthropometry and medical condition, as well as their progression and acquisition of skills and their compliance to the program. At the beginning of the intervention a complete nutritional evaluation was performed and a diet based on age, pubertal stage, and physical activity requirements, according to the World Health Organization and Food and Agricultural Organization guidelines, was prescribed.[16] The recommended diet composition was 55% carbohydrates, 20% proteins, 25% lipids (<7% saturated fat, <300 mg/d cholesterol, and <1% trans fat), and <3 g of salt per day. Participants filled out a 24-hour nutritional recall questionnaire during the 3 days prior to their follow-up appointment to assess diet compliance. All patients were encouraged to participate in sports activities at last 5 days a week and for a minimum of 60 minutes.

To evaluate physical activity compliance, we tested fitness achievement using the Harvard step test modified for the pediatric population and a physical fitness score was calculated;[17] evaluations were applied at baseline and at the postintervention state. The overall intervention compliance was evaluated through anthropometric, metabolic, and fitness parameter modifications, as well as through the acquisition of healthy behavior knowledge.

Clinical Trial Design

This trial was conducted in accordance with the Declaration of Helsinki and adhered to Good Clinical Practice Guidelines issued by the International Conference of Harmonization. The children and their parents provided written informed assent/consent. Eligible patients were included in the lifestyle intervention program (LIP) and randomized to receive either MET (1 g/d), CLA containing 50:50 isomers c9,t11 and t10,c12 (3 g/d), or PLB (1 g/d) 3 times a day for 16 weeks. Visits were scheduled monthly. Diet, exercise, and medication compliance, as well as anthropometric variables, were recorded during each visit. The final evaluation was similar to baseline; EHCT and skeletal muscle biopsies were performed at the postintervention state. Patients were eliminated when they showed poor compliance to medication (<80% or >100%) or intolerance, or when ≥1 workshop sessions were missed.

Anthropometric and Metabolic Evaluation

Baseline evaluation consisted of complete anthropometric and body composition analysis. Height and weight were obtained with participants in light clothes and without shoes, using a standardized stadiometer and mechanical scale. A 12-hour fasting blood sample was drawn. Laboratory measurements included glucose, lipid profile, and aminotransferases that were analyzed enzymatically with the use of commercially available reagents. Insulin was measured using Bio-Plex Pro human diabetes insulin immunoassay by Bio-Rad (Hercules, CA). Fasting insulin resistance and sensitivity surrogated indexes were calculated as follows: homeostatic model assessment of insulin resistance (HOMA-IR) = [fasting plasma insulin (μU/mL) × fasting plasma glucose (mmol/L)]/22.5, and quantitative insulin sensitivity check index (QUICKI) = 1/[log fasting plasma insulin (μU/mL) + log fasting plasma glucose (mg/dL)].

Clamp Procedure

A 2-hour euglycemic-hyperinsulinemic clamp was performed[18] and executed during a 12-hour fasting condition. Intravenous catheters were inserted in the right and left forearm vein, one in a retrograde direction, and warmed in a box that was designed for this purpose (Kepis Keipis One Device, unpublished data). This device allowed the introduction of the complete forearm and maintenance of adequate high temperature and humidity that provided an arteriovenous shunt for blood sample supply while avoiding burns. The additional vein was used to infuse insulin and 20% dextrose solution at variable rates. Intravenous crystalline insulin (Humulin; Eli Lilly & Co., Indianapolis, IN) was used. A priming insulin dose of 120 mIU/m2 of body surface (bs) per minute at time 0 was administered after 1 hour of baseline and during the first 5 minutes. Thereafter, the infusion was gradually reduced to60mIU/m2bs/min up to minute 10 and maintained through the end of the clamp. Glucose infusion started at minute 5 (5 mg/kg/min in all the patients according to information obtained during the standardization procedure). The samples were obtained every 5 minutes, and glucose infusion was dynamically modified to clamp plasma glucose at 85 to 95 mg/dL.

The rate of glucose disposal (Rd) was calculated and adjusted during the last 30 minutes of the clamp when plasma glucose stabilized at a fixed range.

Primary endpoints included the postintervention insulin resistance state defined as the Rd value (mg/kg/min) measured via EHCT, as well as the evaluation of surrogate indexes of insulin resistance and sensitivity (insulinemia, HOMA-IR, and QUICKI). The expression of insulin receptor substrates IRS1, IRS2, and IRS4 in muscle biopsies complemented the insulin resistance study. Secondary objectives were modifications of anthropometric and metabolic parameters. Moreover, medication safety and tolerability were important outcomes.

Muscle Biopsies

The participation of MET and CLA on the insulin signaling pathway was explored with muscles biopsies from the vastus lateralis performed under local anesthesia after 16 weeks of intervention. An incision with a no. 11 surgical blade was made to insert an 8 swg (4.0 mm) Bergstrom needle (Ultramed, Milton, ON, CA).

RNA Isolation

Total RNA was isolated from biopsies samples using an RNeasy fibrous tissue minikit for muscle and an RNeasy lipid tissue minikit for adipose tissue (Qiagen, Valencia, CA) following the manufacturer's protocol. RNA concentration was determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA). Integrity was evaluated by agarose gel electrophoresis using a vertical chamber Enduro (Labnet International, Edison, NJ) and the UltraSlim LED Illuminator SLB-01 (Maestrogen, Las Vegas, NV).

Genetic Expression of Insulin Receptors

The genetic expression patterns of IRS1, IRS2, and IRS4 were studied in 14 and 17 muscular tissue biopsies obtained from the MET and CLA groups, respectively. Quantitative reverse transcription polymerase chain reaction array (human insulin signaling pathway, RT2 Profiler, PAHS-030Z, Qiagen) was performed. Complementary DNA was prepared using an RT2 polymerase chain reaction array first-strand kit (Qiagen) according to the manufacturer's instructions. Normalization was computed with ACTB, B2M, GAPDH, HPRT1, and RPLP0. The expression patterns observed in the MET and CLA groups were compared with muscular tissue samples from the PLB group (n = 17) used as calibrator. The differential gene expression was calculated using the Qiagen software polymerase chain reaction analyzer through the 2−ΔΔCt analysis, and a 2.5-fold change cut-off (P < 0.05) was considered.

Statistical Analysis

Descriptive statistics for all numerical variables are reported as the mean and standard deviation and standard error of the mean (SEM) for contrasts as indicated in the text or figures. Contrast among treatment groups was assessed by analysis of variance and analysis of covariance (ANCOVA) for adjustment by confounding variables. Post hoc analysis and the multiple contrast hypothesis corrected by Fisher's least significant distance were executed. The η2 effect sizes obtained from ANCOVAs were transformed to Cohen's d. χ2 Analyses were also executed to evaluate differences in proportion among groups. SPSS software version 22 (IBM, Armonk, NY) was used to conduct the statistical analyses. A probability of a error of <5% was considered as statistically significant.