Transcriptomics of Mononuclear Cells and Inflammatory Status of Obese Patients Treated With Omega-3 Fatty Acids

Longitudinal Analysis of the Transcriptome From Mononuclear Blood Cells and of the Inflammatory Status of Obese Patients, Supplemented With High Doses of Omega-3 Fatty Acids (EPA and DHA)

The main objective is to determine the effect that supplementation with 4.8 g/day of w-3 FA [3.2g eicosapentaenoic acid (EPA) and 1.6 g docosahexaenoic acid (DHA)] have on the inflammatory state of obese patients (BMI ≥ 35.0 kg/m2), at the metabolic, cellular and molecular levels.

Study Overview

• Status: Withdrawn
• Conditions: Inflammation; Obesity; Immune System Disorder
• Intervention / Treatment: Dietary supplement: Fish oil

Detailed Description

Methodology Study design. This is a prospective, experimental, comparative, non-blinded (open) study.

Adult obese (BMI ≥ 35.0 kg/m2) patients, age 25 to 45 y, of any gender will be recruited from the outpatient Clinic of Obesity at the INCMNSZ. The patients may have (or not) insulin resistance, but must not have any of the following: type 2 diabetes (controlled or not), hypertension, liver or kidney failure, metabolic syndrome, taking antinflammatory drugs or dietary supplements, and in the case of women, pregnant or lactating.

The control group will consist of 40 healthy adult non-obese volunteers (BMI 18.5 to 23.0 kg/m2), age 25 to 45 y, of any gender who accept to participate in the study. This group will only be studied at the beginning (time 0) and they will not consume the fish oil supplement, since its use will be to obtain reference values for all variables described.

Fish oil supplement will be the source of omega-3 fatty acids (w-3 FA) and this will be a donation from the Inflammation Research Foundation (IRF, Boston MA), in the form of sealed unmarked bottles with 120 capsules/bottle.

Each 1 gram capsule contains 0.6 g of w-3 FA (0.4 g of EPA and 0.2 g of DHA). In order to provide 4.8 g of w-3 FA each obese patient must consume 8 capsules/day, distributed as follows: 2 capsules at breakfast, 4 capsules with the main meal and 2 capsules at dinner.

Study design. Each participant (control subjects and obese patients) will receive 2 bottles (240 capsules) of the fish oil at the time of recruitment (time 0) which is enough to cover the dosage for 1 month. After the first month had passed, the remaining capsules (if any) will be counted and 2 new bottles of supplement will be provided to cover the dosage of the following month. This strategy will be repeated at the end of month +2 to cover the dosage of month +3. A "wash-out" period of at least 1 month will be observed (month +4). All variables described (except anthropometry) will be analyzed at times 0, +2 and +4.

Anthropometry. Height in meters (m), weight in kilograms (kg), will be used to calculate the body mass index (BMI) with the formula [weight (kg)/ height (m)2]. Body composition will be determined by electric bioimpedance with a Full Body Composition Analyzer X-Contact 356, to quantify the fat mass (in % and in kg) and the fat-free (lean) mass (in % and kg). Anthropometry will be done only at times 0 and +4.

Blood samples. Venous blood will be obtained at times 0, +1, +2, +3 and +4 with the Vacutainer system with anticoagulant (EDTA-K2). An aliquot of blood will be centrifuged to obtain plasma and stored at -80°C until assayed. Peripheral blood mononuclear cells (PBMNC) will be obtained by density gradient centrifugation (Lymphoprep). These cells will be further processed for the following determinations: i) transcriptional analysis by RNA-seq and qPCR, ii) flow cytometry analysis of immune cells populations (B, T and monocytes), iii) cellular and energy metabolism (Seahorse) and, iv) the "Treg-mediated suppression of cellular proliferation (TMSCP)" assay.

Biochemical analysis. Serum levels of the following metabolites, lipids, cytokines and adipokines will be determined: glucose, insulin, total cholesterol, HDL, LDL, triglycerides, adipokines (leptin, adiponectin, IL-6, TNFalpha, MCP1), and interleukin (IL) 1beta, 2, 4, 8, 10, 12, 17 and TGFbeta. Fatty acid levels will be determined by gas chromatography in serum samples, and in cellular membranes from erythrocytes and leukocytes (PBMNC). The concentration of SPM (resolvins, protectins and maresins) will be determined by HPLC coupled to a mass spectrometer.

Bioenergetics analysis of PBMNC. Cellular metabolism will be analyzed with a Seahorse XFe96 Analyzer (Agilent) to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a 96-well plate format. OCR and ECAR rates are key indicators of mitochondrial respiration and glycolysis respectively. From this analysis of energy consumption, a "bioenergetic health index" (BHI) will be obtained. This BHI reflects the dominant source of energy employed by the cells and can reflect the metabolic pathway employed. For instance, if a high amount of OCR is obtained, this reflects an aerobic metabolism (fatty acid oxidation and oxidative phosporylation, ie, FAO and OXPHOS), whereas a high ECAR indicates anaerobic metabolism and glycolysis is the main metabolic pathway employed.

Transcriptional analysis of gene expression. Total RNA will be obtained from PBMNC and subpopulations (namely, monocytes and Treg cells) using the Trizol reagent and isopropanol precipitation. RNA integrity will be determined in 1% agarose gels by electrophoresis and its concentration by UV spectrophotometry at 260nm.

Samples of RNA with RIN values of 8 and higher will used for sequencing; complementary DNA (cDNA) will be synthesized with reverse-transcriptase and (dT)15 oligonucleotides. The ends of the cDNA fragments will be ligated to sequencing adaptors and amplified by PCR to produce the RNA-Seq library, which will be ready for sequencing. The sequencing method used in the present study will be next generation sequencing (NGS) with the Illumina platform. Validation of changes in the level of transcription determined by RNA-seq analysis, will be determined by quantitative real-time polymerase chain reaction (qPCR) using a RotorGeneQ thermocycler and SybrGreen as label. 18S rRNA will be used as housekeeping gene.

The most powerful use of RNA-Seq is finding differences in gene expression between two or more conditions (e.g., obese vs not obese); this process is called differential expression. The outputs are frequently referred to as differentially expressed genes (DEGs) and these genes can either be up- or down-regulated (i.e., higher or lower in the condition of interest). A cut-off point of 2 fold difference with a p value <0.05 may be used as a screening procedure (although more astringent thresholds may be applied) followed by graphic resources (volcano plots and a heat maps) that will allow identification of transcripts with the largest expression differences and significance. The list of differentially expressed genes will be analyzed using KEGG, IPA and/or Metacore to predict which metabolic/biochemical process differ between the obese and control groups, and after the supplement with fish oil in the obese group.

• Study Type: Interventional
• Phase: Not Applicable

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 20 years to 45 years (Adult)
• Accepts Healthy Volunteers: Yes
• Genders Eligible for Study: All

Description

Inclusion Criteria:

Control group:

• healthy adult non-obese volunteers (BMI 18.5 to 23.0 kg/m2)

Obese group:

• metabolically healthy adult obese (BMI ≥ 35.0 kg/m2) patients,

Exclusion Criteria:

• type 2 diabetes
• hypertension
• liver or kidney failure
• metabolic syndrome
• taking antinflammatory drugs or dietary supplements
• in the case of women, pregnant or lactating

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Non-Randomized
• Interventional Model: Parallel Assignment
• Masking: Single

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: OBESE PATIENTS; Obese patients will be recruited from the outpatient Clinic of Obesity at the INCMNSZ. They will consume the fish oil equivalent to 4.8 g/day of EPA and DHA for 3 months, followed by a one-month period without treatment.	Dietary supplement: Fish oil; They will consume the fish oil equivalent to 4.8 g/day of EPA and DHA for 3 months, followed by a one-month period without treatment.
Active Comparator: CONTROL GROUP; Healthy normal volunteers will be recruited from friends and family of the investigators, and staff at the INCMNSZ. They will consume the fish oil equivalent to 4.8 g/day of EPA and DHA for 3 months, followed by a one-month period without treatment.	Dietary supplement: Fish oil; They will consume the fish oil equivalent to 4.8 g/day of EPA and DHA for 3 months, followed by a one-month period without treatment.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Changes in serum triglycerides levels	A reduction in serum triglycerides of at least 5% of initial concentration	Within the time of intervention, i. e., 3 months

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Changes in serum levels of inflammatory proteins, including cytokines	A reduction in the levels of pro-inflammatory cytokines (IL-6) and adipokines (leptin), and an increase of anti-inflammatory cytokines (IL-10) and adipokines (adiponectin)	Within the time of intervention, i.e., 3 months

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Changes in the Bioenergetic index of PBMNC (monocytes and lymphocytes)	A change in the amount of energy expended by monocytes and lymphocytes after treatment with omega-3 fatty acids (EPA and DHA)	Within the time of intervention, i.e., 3 months

Collaborators and Investigators

• Sponsor: Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran

Study record dates

Study Major Dates

• Study Start (Actual): January 16, 2017
• Primary Completion (Actual): June 1, 2020
• Study Completion (Anticipated): January 1, 2024

Study Registration Dates

• First Submitted: January 21, 2022
• First Submitted That Met QC Criteria: January 21, 2022
• First Posted (Actual): February 2, 2022

Study Record Updates

• Last Update Posted (Actual): March 15, 2023
• Last Update Submitted That Met QC Criteria: March 13, 2023
• Last Verified: January 1, 2023

More Information

Terms related to this study

• Keywords: DHA; omega-3 fatty acids; EPA
• Additional Relevant MeSH Terms: Pathologic Processes; Inflammation; Immune System Diseases
• Other Study ID Numbers: 1832

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: All pertinent data from the participants most likely will be available for their review by Regulatory Authorities, Scientific Committees and other scientific personnel.
• IPD Sharing Time Frame: After completion of the study
• IPD Sharing Access Criteria: None assigned
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; ICF

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No