Chia oil induces browning of white adipose tissue in high-fat diet-induced obese mice

Journal Pre-proof Chia oil induces browning of white adipose tissue in high-fat diet-induced obese mice Thamiris de Souza, Simone Vargas da Silva, Thaís Fonte-Faria, Vany NascimentoSilva, Christina Barja-Fidalgo, Marta Citelli PII:

S0303-7207(20)30072-1

DOI:

https://doi.org/10.1016/j.mce.2020.110772

Reference:

MCE 110772

To appear in:

Molecular and Cellular Endocrinology

Received Date: 29 August 2019 Revised Date:

3 February 2020

Accepted Date: 24 February 2020

Please cite this article as: de Souza, T., Vargas da Silva, S., Fonte-Faria, Thaí., Nascimento-Silva, V., Barja-Fidalgo, C., Citelli, M., Chia oil induces browning of white adipose tissue in high-fat dietinduced obese mice, Molecular and Cellular Endocrinology (2020), doi: https://doi.org/10.1016/ j.mce.2020.110772. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

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Chia oil induces browning of white adipose tissue in high-fat diet-induced obese mice

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Thamiris de Souza1, Simone Vargas da Silva2, Thaís Fonte-Faria2, Vany Nascimento-Silva2, Christina

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Barja-Fidalgo2, Marta Citelli1

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RJ, Brazil

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Institute of Nutrition, Department of Basic and Experimental Nutrition, Rio de Janeiro State University,

Department of Cellular Biology, Rio de Janeiro State University, RJ, Brazil

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Corresponding author: Dr. Marta Citelli, Department of Basic and Experimental Nutrition, Institute of

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Nutrition, Rio de Janeiro State University, Rua São Francisco Xavier, 524, Laboratory of Nutrition

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Biochemistry, Rio de Janeiro, 20550-900, RJ, Brazil Phone/Fax: +552122340679; e-mail:

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[email protected]

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Abstract

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Previous research suggests that omega-3 fatty acids from animal origin may promote the browning of

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subcutaneous white adipose tissue. We evaluated if supplementation with a plant oil (chia, Salvia

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hispanica L.) rich in alpha-linolenic fatty acid (C18:3; ω-3) would promote browning and improve

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glucose metabolism in animals subjected to an obesogenic diet. Swiss male mice (n= 28) were divided

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into 4 groups: C: control diet; H: high-fat diet; HC: animals in the H group supplemented with chia oil

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after reaching obesity; HCW: animals fed since weaning on a high-fat diet supplemented with chia oil.

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Glucose tolerance, inflammatory markers, and expression of genes and proteins involved in the

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browning process were examined. When supplemented since weaning, chia oil improved glucose

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metabolism and promoted the browning process and a healthier phenotype. Results of this study

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suggested that chia oil has potential to protect against the development of obesity-related diseases.

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Keywords: browning; chia oil; obesity, alpha-linolenic acid

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1. INTRODUCTION

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Obesity is an important public health problem due to the severity of its associated comorbidities

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and its high prevalence. This condition is characterized by the excessive accumulation of fat in adipose

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tissue (World Health Organization, 2016). The different types of adipose tissue have important

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implications for the pathogenesis and treatment of metabolic complications related to obesity. There are

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two types of adipose tissue classified depending on embryonic origin: white adipose tissue (WAT) and

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brown adipose tissue (BAT) (Luo and Liu 2016). WAT plays a key role in endocrine and metabolic

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functions, as it participates in regulating energy homeostasis and insulin sensitivity (Rosen and

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Spiegelman, 2014). BAT contains multiple mitochondria and its main function to maintain the central

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body temperature in response to cold is regulated by uncoupling-1 protein (UCP-1) (Cannon and

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Nedergaard, 2004).

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Due to the increase in the number of mitochondria and to the acquisition of phenotypic

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characteristics similar to those of BAT, adipocytes in the subcutaneous adipose tissue have been

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described as beige adipocytes when they undergo a process called browning (Cannon and Nedergaard,

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2004; Petrovic et al., 2010). In response to cold stress and treatment with agonists of peroxisome

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proliferator-activated receptor-gamma (PPAR-γ) or β-adrenergic receptor, the development of beige

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adipocytes is increased (Li et al., 2014; Ye et al., 2013). An abundance of beige adipocytes in white

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adipose tissue results in improved glucose metabolism and obesity-related insulin resistance (Sidossis,

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and Kajimura, 2015; Stanford et al., 2015). In addition, identifying new regulators that promote adipose

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tissue energy expenditure and activity to oxidize metabolic substrates can provide key therapeutic

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targets in obesity, diabetes, and dyslipidemia. Studies have shown that some dietary molecules may be

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effective in potentiating BAT thermogenic functions and improving metabolism (Okla et al., 2017).

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Some previous studies suggested that omega-3 polyunsaturated fatty acids from animal origin may

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improve the remodeling and browning in subcutaneous WAT and thermogenic markers in BAT in mice

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(Laiglesia et al., 2016; Lund et al., 2018; Bargut et al., 2016). However, the potential of a plant-derived

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omega-3 fatty acid (alpha-linolenic acid; ALA; C18: 3 n-3) to induce the browning process needs to be

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investigated.

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Chia (Salvia hispanica L.) is an oleaginous seed whose oil is the richest known source of

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omega-3 fatty acids (Cahill, 2003). On average, it contains about 64% ALA (Ayerza et al., 2002;

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Álvarez-Chávez et al., 2008; Mohd Ali et al., 2012). In vivo and in vitro studies have confirmed the

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beneficial effects of omega-3 fatty acid supplementation on lipid and glucose metabolism (Wang and

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Huang, 2015; Fonte-Faria et al., 2019).

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Previously, our group observed that chia oil supplementation in obese mice was able to reverse

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metabolic syndrome by increasing the efficiency of insulin signaling, while also increasing lean mass

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and reducing fat mass (Fonte-Faria et al., 2019). Therefore, considering the content of omega-3 fatty

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acids present in chia oil, as well as their benefits to glycemic homeostasis, we aimed to test the

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hypothesis that chia oil could potentially induce the browning process in subcutaneous WAT.

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MATERIAL AND METHODS

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2.1. Animals and diet protocol

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The Animal Experimentation Ethics Committee of Rio de Janeiro State University approved the animal

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procedures (authorization number: CEA 047/2013). Protocols were carried out in strict accordance with

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the ethical standards of the Declaration of Helsinki of 1964. Swiss mice were obtained from animal

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facilities of the Federal University of Minas Gerais (UFMG, Belo Horizonte, Brazil). The animals were

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housed under controlled conditions of a 12-hour light/dark cycle, 60% humidity, ambient temperature of

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(23 ± 1 °C), and free access to food and water. Male mice were divided according to diet: the control

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group (n = 7) received a normocaloric diet (13% energy derived from fat) and group H received a high-

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fat diet (45% energy derived from fat) for 130 days. The other two high-fat diet groups were

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supplemented with chia oil (1.5% w/w) from day 21 to 130 (HCW) or from day 90 to 130 (HC). Details

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of the diets are provided in a previously published study (Fonte-Faria et al., 2019). The complete

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chemical composition of the chia oil has already been described by da Silva et al. (2016). Alpha

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linolenic acid constitutes ~60% of its total fatty acids. The study published by Oh et al. (2010) was used

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to define the length of time to be adopted while treating with chia oil, as well as to define the amount of

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oil to be supplemented. Considering the total amount of omega-3 fatty acids used in the study by Oh et

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al. (2010) as well as the fatty acid composition of chia oil, we established an intervention model using

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chia oil to treat mice that became obese following the dietetic protocol of Silva et al. (2016). Chia oil

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was included in the chow. Body weight, food intake, and caloric intake were measured during the

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treatment period. Food consumption was measured by the amount that was left in each cage.

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2.2 Intraperitoneal Glucose Tolerance Test

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Prior to the glucose tolerance test, animals were fasted overnight for 10 ± 2 h and wrapped in a towel the

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following morning to minimize stress. The baseline blood glucose measurements were performed using

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a drop of tail blood. Subsequently, the animals received a glucose challenge (1 g/kg of body weight, i.p.)

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followed by repeated sampling of blood glucose readings at 30, 60, 120, and 150 min. The glucose

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measurements were taken using a handheld glucometer (Accu-Chek Roche®; Indianapolis, IN, USA).

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2.3 Sample Collection

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After completing each experiment, mice were euthanized by with drawing blood from the heart under

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anesthesia with a mixture of ketamine (50 mg/kg) andxylazine (20 mg/kg). Subcutaneous adipose tissue

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from the abdominal region was collected and transferred to tubes, snap frozen in liquid nitrogen, and

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stored at −80 °C until RNA extraction, western blotting, and immunohistochemistry analysis.

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2.4 Measurement of plasma cytokines

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Serum levels of leptin, Tumor Necrosis Factor-alpha (TNF-α), Interleukin-6 (IL6), and Interleukin-10

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(IL10)were measured using appropriate ELISA kits (Peprotech, Rocky Hill, NJ, USA; and Cayman

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Chemical, Ann Arbor, MI, USA; respectively), following the manufacturer’s instructions.

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2.4 Quantification of mRNA in adipose tissue

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qRT-PCR was used for mRNA quantification and the primers used (GenOne Biotechnologies) shown in

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Table 1 were used. Total adipose tissue RNA was extracted with the RNeasy Mini Kit (Qiagen, Hilden,

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Germany) following the manufacturer's recommendations. Total RNA samples were initially quantified

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on a BioDrop µLITE spectrophotometer by analyzing the absorbance at 260nm. The purity of the

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extracted RNA was evaluated by measuring the A260/A280 ratio presented by the equipment software.

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cDNA synthesis was performed from 1 µg of total RNA usinga High Capacity RNA-to-cDNA Kit

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(Applied Biosystems, Foster City, CA, USA), according to the manufacturer's recommendations. The

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samples were incubated at 37 °C for 60 minutes, then at 95 °C for 5 minutes. The cDNA synthesis

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reaction was performed on a Veriti Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Real-

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time PCR analysis was performed on a 7500 Real-Time PCR Applied Biosystems (Applied Biosystems,

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Foster City, CA, USA) instrument by measuring green fluorescence from SYBR Green to quantify the

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amplicons. Standard PCR conditions were 95 °C for 5 minutes and 30 cycles at 95 °C (5 s) and 60 °C

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(10 s), followed by generating a dissociation curve to identify the number of amplicons.

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2.5 Quantification of miRNAs in adipose tissue

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RNA was isolated from the subcutaneous adipose tissue usingan RNeasy Mini Kit Assay (Qiagen,

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Hilden, Germany). MiRNA levels were measure using the Applied Biosystems 7500 Fast Real-Time

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PCR System and the TaqMan MicroRNA Assay kit (Applied Biosystems, Foster City, CA, USA) after

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performing reverse transcription with the TaqMan Transcription Reversion MicroRNA kit (Applied

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Biosystems, Foster City, CA, USA). The reactions were performedin 96-well plates (MicroAmp Optical

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96-Well Reaction Plate, Applied Biosystems, Foster City, CA, USA). The TaqMan assays used were

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miRNAs 30b (mmu481432 miR), 196a (mmu481627 miR) and 494 (mmu478135 miR), according to the

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manufacturer's instructions. U6 small RNA was used as an endogenous control for miRNA expression

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analysis.

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2.5 Immunohistochemistry

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For immunohistochemistry, adipose tissues from animals were fixed in paraformaldehyde (4% w/v); and

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paraformaldehyde (4% w/v) with sucrose (10% w/v) for 30 minutes in each solution. The tissues were

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stored in phosphate buffered saline (PBS) with 20% (w/v) sucrose at 4°C until use. For the inclusion of

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the tissue, the samples were embedded in paraffin and cut with a microtome. Then, the slides were

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dewaxed in xylol and ethanol solution (1:1) three times for three minutes and hydrated in decreasing

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concentrations of ethyl alcohol (95%, 70%, and 50%). Then, slideswere washed 4 times in PBS,

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blocked, and permeabilized with PBS/bovine serum albumin (BSA) containing 5%/Triton-X 0.3%

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solution for 1 hour, incubated with a specific anti-UCP1 primary antibody (D9D6X, Rabbit mAb

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#14670, Cell Signaling Technology, Danvers, Massachusetts, EUA) or a DAPI primary antibody

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(Sigma-Aldrich, St. Louis, Missouri, EUA) overnight. The slides were then washed six times with PBS

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and incubated with specific secondary antibodies for 1 hour. The images were captured at 20X and 40X

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magnification on an Olympus BX40 fluorescence microscope. Secondary antibodies for the respective

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primary antibodies were used as negative controls. The images were scanned and processed using

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ImageJ and Adobe Photoshop software.

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2.6 Western blotting

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Western blotting was performed with samples of subcutaneous adipose tissue. The protein concentration

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was determined by using the BCA Protein Assay kit (Pierce Biotech, Rockford, USA). The samples

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were then treated with 5X concentrated sample buffer (50 mM Tris HCl, pH 6.8, 1% SDS, 5% β-

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mercaptoethanol, 20% glycerol, 0.001% bromophenol blue) for 5 minutes at 95 °C and then frozen.

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Samples containing 20 µg of protein were separated by 10% SDS-containing polyacrylamide gel

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electrophoresis (SDS-PAGE). A molecular weight standard was used to estimate protein sizein all gels

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(Rainbow Molecular Weight Marker, Amersham Biosciences). The proteins were transferred to PVDF

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membranes (Hybond P PVDF, Amersham Pharmacia Biotech) for 30 minutes using asemi-dry system

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(BIO RAD, Hercules, CA, EUA). The membranes were incubated for 1 hour with blocking solution

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containing 5% bovine serum albumin (BSA;Sigma-Aldrich, St. Louis, Missouri, EUA) and T-TBS

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(0.1% Tween 20 in TBS), then incubated at 4 °C with rabbit anti-VDAC2 (catalog number: 47104;

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1:500, Abcam, Cambridge, UK) or

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Cambridge, UK). The membranes were washed with T-TBS and then incubated with the specific

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secondary antibody conjugated to biotin (1:5000 1:10000, Santa Cruz Biotechnology, Dallas, Texas,

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EUA) for 1 hour. The membranes were washed with T-TBS and incubated with peroxidase-conjugated

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streptavidin (1:10,000, Zymed, San Francisco, California, USA) for 1 hour. Immunoreactive proteins

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were visualized by ECL labeling using anECL Plus kit (Amersham Biosciences, Pittsburgh, PA) and

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analyzed by ChemiDoc (Bio-Rad, Hercules, CA, EUA). Bands were quantified by densitometry using

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Image J software (NIH, USA).

mouse anti-Actin (catalog number: 101173, 1:1000, Abcam,

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2.7 Statistical analyses

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The results were analyzed statistically using Kruskal–Wallis one-way analysis of variance. The results

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were presented as mean ± standard error (SEM). Values of p <0.05 were considered to indicate statistical

7 172

significance. Data were analyzed using GraphPad Prism version 5.00 for Windows (GraphPad Software,

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La Jolla, CA, USA).

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2.

RESULTS

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3.1 Characterization of the obesity model with chia oil supplementation

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The high-fat diet promoted an increased percentage of body weight gain in animals when compared with

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the control diet (Fig. 1A and 1B). Chia oil supplementation reduced the percentage of body weight gain

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of animals only when presented after obesity was established (Fig. 1B). Taking the period between the

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90th and 130th days of life as a basis, the energy intake of the HC group did not differ from the H group.

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On the other hand, HCW group energy intake was significantly lower (Fig. 1C and 1D).

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3.2 Chia oil supplementation changes the plasma concentration of leptin in obese mice

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The plasma concentration of leptin was reduced in the group supplemented with chia oil since weaning

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(HCW) when compared to the concentration in group H (Figure 2A), although the model of obesity used

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in this study did not cause an increase in circulating leptin levels compared to levels in control. Plasma

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concentrations of TNF-α, IL-6, and IL-10 did not differ among groups (Fig 2, B–D).

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3.3 Chia oil supplementation improves glycemic response

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The supplemented groups (HC and HCW) exhibited greater efficiency of glucose uptake since they

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presented lower glycemia throughout the glucose tolerance test (Fig. 3A and C). Mice in the high-fat diet

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(H) group exhibited increased fasting blood glucose. On the other hand, supplementation with chia oil

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since weaning (HCW), reduced plasma fasting glucose levels. However, the same did not occur when

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chia oil supplementation was initiated at 90 days of life, when obesity was already established (HC

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group) (Fig. 3B).

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3.4 Chia oil promotes browning of subcutaneous adipose tissue

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The quantification of PPAR-ɤ mRNA in subcutaneous adipose tissue was higher in the groups receiving

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chia oil (Fig. 4A). PGC1-α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) is a

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transcriptional regulator that co-induces mitochondrial biogenesis by activating different transcription

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factors. The groups that received chia oil exhibited increased expression of PGC 1-α mRNA (Fig. 4B).

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UCP-1 is a protein found primarily in the mitochondria of brown adipose tissue and the browning

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process usually increases its expression. In both chia oil-supplemented groups, UCP-1 mRNA

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expression was higher when compared to that of group H (Fig. 4C). mTFA is a mitochondrial

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transcription factor that also participates in mitochondrial genome replication. By using Kruskal–Wallis

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one-way analysis of variance, we did not find any difference among the groups when evaluating mTFA

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mRNA expression (Fig. 4D). However, when comparing pairs of groups instead of comparing all of

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them (Mann–Whitney U test), we observed that the HC and HCW groups showed greater expression

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than the H group (p<0.001), data not shown). HCW group exhibited greater levels of OPA-1 (Fig. 4E),

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which is a gene encoding a protein that regulates mitochondrial fusion. We evaluated the expression of

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carnitine palmitoyltransferase I (CPT-1), a mitochondrial marker and a key enzyme essential to the beta-

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oxidation of long chain fatty acids, but we did not find any difference among the groups (Fig. 4F).

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Subsequently, we quantified levels of miRNAs that are related to mitochondrial biogenesis process,

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miR-196a, miR-30 and miR-494. No differences were observed among the groups (Fig. 4G, H, I).

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Additionally, we analyzed the expression of the VDAC2 protein, which is a mitochondrial channel-

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forming protein (porin) involved in cellular energy metabolism. Elevated VDAC2 expression was

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observed in the HCW group (Fig. 5). The presence of UCP-1 in subcutaneous adipose tissue was also

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observed by immunohistochemistry (Fig. 6). This result indicated a greater browning process in both

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groups receiving chia oil, with a greater effect in the group that received chia oil since weaning.

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4. Discussion

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We identified molecular changes in the subcutaneous adipose tissue of obese animals promoted

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by a plant oil supplementation. Regular ingestion of chia oil, the richest plant source of omega-3 fatty

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acid, promoted the browning process. Browning of subcutaneous adipose tissue has been studied and

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understood as a process capable of attenuating some of the effects of obesity (Langin, 2010). Here, we

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identified increased expression of genes involved in mitochondrial biogenesis in subcutaneous adipose

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tissue. Consistently, there was increased mitochondrial loading, as indicated by increased expression

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of UCP-1 and VDAC proteins. EPA and DHA were previously associated with increased expression

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of UCPs in multiple tissues, such as white and brown adipose tissues, liver, and muscle (Sadurskis et

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al., 1995; Oudart et al., 1997; Baillie et al., 1999; Armstrong and Towle, 2001; Zhao and Chen, 2014).

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Similar to the results of the present study, the Marineli et al. (2015) study showed that

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prolonged treatment with chia seeds and short treatment with chia oil in obese animals restored the

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expression of PGC1-α. In addition, treatment with fenofibrate (a PPAR-α agonist) is capable of

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enhancing PGC1-α expression in the subcutaneous adipose tissue of animals, suggesting a central role

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that this transcriptional regulator of mitochondrial biogenesis plays in the browning process (Rachid et

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al., 2015).

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On the other hand, we observed that supplementation with chia oil did not alter the expression

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of miR-196a and miR-30, potential inducers of brown adipogenesis in white adipose tissue. They

9 244

represent promising targets for modulating the complex pathways and as potential regulators of

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adipose tissue form and function, and the transcriptional programs that respond to ‘stress’ in adipose

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tissue in obesity. MiR-196a seems to induce brown adipogenesis in human WAT and is upregulated in

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inguinal WAT from mice exposed to cold or adrenergic stimulation (Mori et al., 2012; Schimanski et

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al., 2009). Mice overexpressing miR-196a showed resistance to obesity and improved glucose

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metabolism (Mori et al., 2012). Concurrently, the role of miR-30 in regulating thermogenesis was

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previously described in vitro and in vivo. Overexpression of miR-30 greatly increased thermogenic

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gene expression in primary subcutaneous white adipocytes (Hu et al., 2015). Considering the

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involvement of miR 196 and 30 in the browning process, we evaluated these markers after

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supplementation with chia oil. Additionally, we also evaluated the expression of miR-494 in the

254

adipose tissue since it is known to regulate mitochondrial biogenesis by downregulating mt-TFA in

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skeletal muscle tissue (Yamamoto et al., 2012). It seems that these miR are not involved in the

256

browning process observed in the present study.

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Here we showed that supplementation with chia oil after the establishment of obesity (HC group),

258

decreased the percentage of body weight gain. It has already been observed that omega-3 fatty acid

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intake decreases the risk of body weight gain (Fonte-Faria et al., 2019). In contrast, when supplemented

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with chia oil since weaning (HCW group), did not reduce the body weight gain. In the present study,

261

changes in body composition were not evaluated. However, it is possible that the supplementation

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performed from weaning increased the percentage of lean mass, so that the body weight was not

263

changed overall. A previous study by our group found that supplementation with chia oil promoted an

264

increase in lean mass and decreased fat mass (Fonte-Faria et al., 2019).

265

The browning of WAT impact energy expenditure and glucose homeostasis. GPR120, a lipid-

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sensitive G protein-coupled receptor (GPCR), may function as the omega-3 fatty acid receptor that

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mediates its anti-inflammatory and insulin sensitizing effects. In addition, GPR120 has recently been

268

reported to influence BAT activation, especially the browning of subcutaneous WAT (Quesada-López

269

et al., 2016). Dietary enrichment with omega-3 PUFAs has beneficial effects on metabolic health in

270

healthy subjects and in individuals with metabolic disorders (Takahashi and Ide, 2000; Flachs et al.,

271

2011; Villarroya et al., 2014).

272

Here, plasma leptin levels were reduced in the HCW group, consistently with reduced energy

273

intake observed from the 90th to the 130th (Fig 1D). Surprisingly, animals fed a high-fat diet (H group)

274

did not show an increase in serum leptin concentrations, despite their weight gain. In our previous study

275

with a similar design (Fonte-Faria et al., 2019), using C57BL/6 instead of Swiss mice, we found an

276

increase in leptin levels in H group. Moreover, we found decreased leptin levels in HC group compared

277

to H group. Seeger and Murphy (2016) demonstrated the existence of differences in tissue fatty acid

278

uptake and trafficking between C57BL/6 and Swiss strains, highlighting its importance when carrying

279

out fatty acid metabolic studies.

280

Sundaram et al. (2016) compared high-fat diets with different amounts and sources of lipids and

281

observed that diets containing fish oil reduced the plasma concentration of leptin in mice. In humans,

10 282

dietary supplementation with EPA/DHA showed a negative correlation with plasma leptin

283

concentrations (Reseland et al., 2001). Furthermore, it has been described that one of the possible

284

benefits of omega-3 fatty acids is that they reduce levels of inflammatory adipokines, such as leptin.

285

However, when we investigated the concentrations of IL-10, an anti-inflammatory cytokine, we did not

286

identify any differences caused by chia oil supplementation. In addition, no changes were observed in

287

plasma concentrations of IL-6 or TNF-α. These data corroborate new studies that have been

288

questioning the association between inflammation and obesity. A study published by Kim et al. (2015)

289

showed that increased adipocyte size might lead to insulin resistance, regardless of inflammation. In

290

mice treated with clodronate (to deplete macrophages), a high-fat diet induced adipocyte hypertrophy

291

and caused insulin resistance independently of the activation of inflammatory response (Lee et al.,

292

2011).

293

The association of changes in glucose metabolism in response to excessive weight gain has

294

been widely discussed in the literature. Previously, we described changes in the insulin-signaling

295

pathway in response to chia oil supplementation (Fonte-Faria et al., 2019). In the present study,

296

animals supplemented with chia oil presented an improved glycemic response compared to animals

297

that did not receive chia oil.

298

Data from this study indicate that supplementation with chia oil induced the browning process

299

and was even more beneficial to health when supplemented from the beginning of obesity

300

development. Insulin resistance and diabetes mellitus are the main comorbidities of obese individuals.

301

Therefore, the data reinforce the benefits of using chia seed oil to prevent them.

302 303

Conflicts of interest: All authors stated that there were no conflicts of interest.

304 305

Acknowledgments: This study was funded by Fundação Carlos Chagas Filho de Amparo à Pesquisa do

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Estado do Rio de Janeiro (FAPERJ; grants #E-26/010001754/2015, #E-26/202782/2017), Conselho

307

Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant #408401/2017-6). The authors

308

thank Genilson Silva for technical assistance.

309 310 311

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422 423

Figure captions

424

Figure 1: Body weight curve (A), Weight gain percentage (B), Energy intake (C), Energy intake from

425

90th to 130th day of life (D). C: control diet group (n = 7); H: high fat diet group (n = 7); HC: high-fat

426

diet supplemented with chia oil (n = 7); HCW: high-fat diet supplemented with chia oil since weaning (n

15 427

= 7). The arrows indicate the beginning of chia oil supplementation. The Kruskal–Wallis one-way

428

analysis of variance was used for statistical analysis. Data are expressed as mean ± SD and statistically

429

significant differences are set at P <0.05.

430 431

Figure 2: Plasma levels of leptin (A), TNF-alpha (B), IL-6 (C), and IL-10 (D) were analyzed by ELISA.

432

C: control diet group (n = 7); H: high fat diet group (n = 7); HC: high-fat diet supplemented with chia oil

433

(n = 7); HCW: high-fat diet supplemented with chia oil since weaning (n = 7). The Kruskal–Wallis one-

434

way analysis of variance was used for statistical analysis. Significance was considered for values of

435

P<0.05.

436 437

Figure 3: Glucose tolerance test (GTT) (A), fasting glycemia (B), and area under the GTT curve (C). C:

438

control diet group (n = 7); H: high-fat diet group (n = 7); HC: high-fat diet supplemented with chia oil (n

439

= 7); HCW: high-fat diet supplemented with chia oil since weaning (n = 7). The Kruskal–Wallis one-

440

way analysis of variance was used for statistical analysis. Significance was considered for values of

441

P<0.05.

442 443

Figure 4: Evaluation of mRNA levelsof genes encoding PPAR-ɣ (A), PGC-1α (B), UCP-1 (C), mTFA

444

(D), OPA1 (E), and CPT1a (F). Evaluation of expression for miR-196a (G), miR-30a (H) and miR-494

445

(I). C: control diet group (n = 5); H: high-fat diet group (n = 5); HC: high-fat diet supplemented with

446

chia oil (n = 5); HCW: high-fat diet supplemented with chia oil since weaning (n = 5). The Kruskal–

447

Wallis one-way analysis of variance was used for statistical analysis. Significance was considered for

448

values of P<0.05.

449 450

Figure 5: Western blot image of VDAC2 expression normalized to actin expression. C: control diet

451

group (n = 7); H: high-fat diet group (n = 7); HC: high-fat diet supplemented with chia oil (n = 7); HCW:

452

high-fat diet supplemented with chia oil since weaning (n = 7). Data are expressed as mean ± SD and

453

statistically significant differences are set at P <0.05. The Kruskal–Wallis one-way analysis of variance

454

was used for statistical analysis.

16 455

Figure 6: Immunohistochemical image with UCP-1 labeling. C: control diet group (n = 5); H: high-fat

456

diet group (n = 5); HC: high-fat diet supplemented with chia oil (n = 5); HCW: high-fat diet

457

supplemented with chia oil since weaning (n = 5).

458

Table 1. List of primers used for quantitative real-time PCR analysis. Gene

Forward (5' to 3')

Reverse (5' to 3')

GAPDH

ACAATGAATACGGCTACAGCAACAG

GGTGGTCCAGGGTTTCTTACTCC

PPARγ

GAGTGTGACGACAAGATTTG

GGTGGGCCAGAATGGCATCT

PGC1-α

AAGTGTGGAACTCTCTGGAACTG

GGGTTATCTTGGTTGGCTTTATG

UCP-1

AGATCTTCTCAGCCGGAGTTT

CTGTACAGTTTCGGCAATCCT

OPA-1

TGGAAAATGGTTCGAGAGTCAG

mTFA

CACCCAGATGCAAAACTTTCAG

CATTCCGTCTCTAGGTTAAAGC G CTGCTCTTTATACTTGCTCACAG

CPT1a

CTCAGTGGGAGCGACTCTTCA

GGCCTCTGTGGTACACGACAA

Table 1. Sequences of real-time PCR primers. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PPAR-γ: peroxisome proliferator-activated receptor-gamma; PGC-1α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; UCP-1: uncoupling-1 protein; OPA-1: mitochondrial dynamin like GTPase; mTFA: mithocondrial transcription factor; CPT1a: carnitinepalmitoyltransferase 1A

Highlights

Chia oil supplementation increases browning of subcutaneous adipose tissue. Obese animals treated with chia oil supplementation improves glucose metabolism. Chia oil supplementation is effective in treating obesity.