The roles of triiodothyronine and irisin in improving the lipid profile and directing the browning of human adipose subcutaneous cells

The roles of triiodothyronine and irisin in improving the lipid profile and directing the browning of human adipose subcutaneous cells

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Journal Pre-proof The roles of triiodothyronine and irisin in improving the lipid profile and directing the browning of human adipose subcutaneous cells Miriane de Oliveira, Lucas Solla Mathias, Bruna Moretto Rodrigues, Bianca Gonçalves Mariani, Jones Bernardes Graceli, Maria Teresa De Sibio, Regiane Marques Castro Olimpio, Fernanda Cristina Fontes Moretto, Igor Carvalho Deprá, Célia Regina Nogueira PII:

S0303-7207(20)30044-7

DOI:

https://doi.org/10.1016/j.mce.2020.110744

Reference:

MCE 110744

To appear in:

Molecular and Cellular Endocrinology

Received Date: 28 June 2019 Revised Date:

28 January 2020

Accepted Date: 28 January 2020

Please cite this article as: de Oliveira, M., Mathias, L.S., Rodrigues, B.M., Mariani, Bianca.Gonç., Graceli, J.B., De Sibio, M.T., Castro Olimpio, R.M., Fontes Moretto, F.C., Deprá, I.C., Nogueira, Cé.Regina., The roles of triiodothyronine and irisin in improving the lipid profile and directing the browning of human adipose subcutaneous cells, Molecular and Cellular Endocrinology (2020), doi: https://doi.org/10.1016/j.mce.2020.110744. 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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The roles of triiodothyronine and irisin in improving the lipid profile and directing the

2

browning of human adipose subcutaneous cells

3

Miriane de Oliveira1*, Lucas Solla Mathias1, Bruna Moretto Rodrigues1, Bianca Gonçalves

4

Mariani1, Jones Bernardes Graceli2, Maria Teresa De Sibio1, Regiane Marques Castro Olimpio1,

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Fernanda Cristina Fontes Moretto1, Igor Carvalho Deprá1, Célia Regina Nogueira1

6

1

7

Botucatu, São Paulo, Brazil

8

2

9

*

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Department of Internal Clinic, Botucatu Medicine School, São Paulo State University (UNESP),

Department of Morphology, Federal University of Espírito Santo, Espírito Santo, Brazil

Correspondence: [email protected]; São Paulo State University Julio de Mesquita

Filho Botucatu Campus Faculty of Medicine

11 12

Abstract

13

Triiodothyronine (T3) and irisin (I) can modulate metabolic status, increase heat production, and

14

promote differentiation of white adipose tissue (WAT) into brown adipose tissue (BAT). Herein,

15

human subcutaneous white adipocytes were treated with 10 nM T3 or 20 nM I for 24 h to evaluate

16

intracellular lipid accumulation, triglyceride, and glycerol levels, oxidative stress, DNA damage,

17

and protein levels of uncoupling protein 1 (UCP1), adiponectin, leptin, peroxisome proliferator-

18

activated receptor gamma (PPARγ), and fibronectin type III domain-containing protein 5 (FNDC5).

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T3 and irisin improved UCP1 production, lipid profile, oxidative stress, and DNA damage. T3

20

elevated adiponectin and leptin levels with a concomitant decrease in PPARy and FNDC5 levels.

21

However, irisin did not alter adipokine, PPARy, and FNDC5 levels. The results indicate that T3 may

22

be used to increase leptin and adiponectin levels to improve insulin sensitivity, and irisin may be

23

used to prevent obesity or maintain weight due to its impact on the lipid profile without altering

24

adipokine levels.

25 26

Keywords: oxidative stress, fat, thyroid hormone, lipid accumulation, RNA-seq

27 28

Highlights:

29

1. T3 and irisin decreased intracellular lipid accumulation and cellular damage.

30

2. T3 and irisin up-regulated UCP1 levels in adipocytes.

31

3. T3 stimulated the production of adiponectin and leptin.

32

4. Irisin maintained the levels of adiponectin and leptin.

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5. T3 decreased the levels of FNDC5 and PPARγ.

34 35 1

36

Introduction

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Adipose tissue (AT) can be classified into two main types: white adipose tissue (WAT) and

38

brown adipose tissue (BAT). WAT acts as the primary site for energy storage, while BAT is involved

39

in thermogenesis and maintains body temperature in cold conditions. Recently, both AT types have

40

been shown to play critical endocrine roles through the production and secretion of adipokines such

41

as leptin and adiponectin [1]. Abnormal metabolic conditions could result from a sustained positive

42

energy balance (when the energy intake is higher than the energy expenditure), and exacerbated

43

body weight gain is associated with irregular fat accumulation, which leads to obesity [2-4].

44

The incidence of obesity and associated diseases is increasing worldwide. Complications

45

associated with obesity include insulin resistance, irregular lipid profile, DNA damage and

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oxidative stress [5-8], abnormal levels of leptin and adiponectin, and other metabolic disorders [3].

47

Previous studies have investigated various approaches, including limiting food intake, exercise,

48

and/or the use of thermogenic compounds like thyroid hormones (THs), to treat or reduce

49

complications associated with obesity [9].

50

The thyroid synthetizes and releases THs, triiodothyronine (T3), and thyroxine (T4). These

51

are iodine‐containing compounds derived from tyrosine. They exert biological effects by interacting

52

with their cognate nuclear receptors. T4 is the major product of the thyroid, but T3 is the most

53

active of thyroid products. T3 controls several critical biological processes and provides a negative

54

feedback for the synthesis of thyrotropin‐releasing hormone (TRH) by the hypothalamus and

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thyrotropin (also known as thyroid‐stimulating hormone, TSH) by the pituitary gland [10]. T3 acts

56

through its nuclear receptors or by activating extranuclear pathways to modulate several

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physiological functions and maintain a normal metabolism [11, 12].

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THs influence key metabolic pathways that control energy balance by regulating energy

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storage and expenditure, and modulating thermogenesis in AT [13-15]. Hyperthyroidism, which

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occurs when THs are excessively produced, promotes a hypermetabolic state characterized by

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increased resting energy expenditure, weight loss, reduced cholesterol levels, increased lipolysis,

62

and increased gluconeogenesis [15]. Conversely, hypothyroidism, which is the consequence of

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reduced TH levels, is associated with a hypometabolic state characterized by reduced resting energy

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expenditure, weight gain, increased cholesterol levels, reduced lipolysis, and reduced

65

gluconeogenesis [15].

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Recently, skeletal muscle has been identified as an extremely active endocrine organ that

67

secretes a great variety of cytokines and other paracrine factors. They include irisin and are

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collectively termed myokines. These have been proposed as mediators of the beneficial actions of 2

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physical activity and regulators of the metabolic function in AT [16]. Irisin is a soluble muscle-

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specific peptide that is released into the circulation during the proteolysis of fibronectin type III

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domain-containing protein 5 (FNDC5) [17].

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In the skeletal muscle of humans and rats, FNDC5 expression is stimulated by peroxisome

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proliferator-activated receptor gamma (PPARγ) and PPARγ coactivator 1-alpha (PGC1-α) after

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exercise [18], which in turn stimulates the expression of uncoupling protein 1 (UCP1) in the

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mitochondria. UCP1 activation inhibits the synthesis of adenosine triphosphate (ATP) and acts as a

76

decoupling protein. Consequently, UCP1 leads to the release of heat [19, 20], thereby decreasing fat

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deposits.

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BAT contributes to body temperature regulation in infants [21] and plays a role in adult

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physiology [17]. Irisin promotes the differentiation of WAT to BAT through a mechanism called

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browning. Expression of BAT genes, particularly UCP1, have been reported in human adult

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abdominal and subcutaneous WAT [22, 23]. In addition, previous studies have reported that forced

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expression of UCP1 in WAT cells results in darkening in vitro [24-27]. In rodent models, the

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browning of WAT seems to have anti-obesity and anti-diabetic effects [28]. In obese subjects,

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FNDC5 levels in the skeletal muscle and circulation are reduced and correlate with decreased

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sensitivity to insulin [29].

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In mice, the exogenous administration of irisin induces the browning of subcutaneous fat

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and stimulates thermogenesis, thereby promoting oxygen consumption [17]. In humans, a positive

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correlation between circulating irisin and energy expenditure has also been found, as the

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expressions of fat browning-specific genes (Ucp1, Pgc1a, Tmem26, Ebf3, Elovl3, Cidea, and

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Cox7a) are mediated by the activation of p38 mitogen-activated protein kinase and extracellular

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signal-regulated kinase (ERK)1/2 pathways [30-32].

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THs and irisin have profound influences on thermogenesis and metabolism in the human

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body. Both T3 and irisin increase heat production and control the energy balance. Comparisons of

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the effects of THs and irisin are scarce. Thus, we aimed to carry out such an evaluation in AT. We

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identified that T3 and irisin could up-regulate UCP1, a marker of the browning of white adipocytes,

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and decrease lipid accumulation, oxidative stress, and DNA damage. Irisin treatment stimulated

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lipolysis as evidence by increases in triglyceride (TG) and glycerol levels. Treatment with T3

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probably led to the oxidation of intracellular fatty acids, which may occur in browning human

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adipocytes. Additionally, we demonstrated that the synthesis of FNDC5 in human subcutaneous

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adipocytes was suppressed by T3. Adipokines, adiponectin, leptin, and PPARγ were maintained at

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basal levels by irisin. However, T3 elevated the levels of adipokines and concomitantly decreased

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PPARγ levels. We also analyzed RNA-seq data to complement our findings and to evaluate the 3

103

effects of T3 and irisin on biological processes associated with mitochondrial activity, lipid droplet

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release, and RNA polymerase II activity.

105 106

Materials and Methods

107 108

Culture and cell differentiation

109 110

Human subcutaneous preadipocytes (HPAd - 802S-05A; Cell Applications, Inc., San Diego,

111

CA, USA) were purchased through Sigma-Aldrich (St. Louis, MO, USA). Preadipocytes were

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cultured in preadipocyte basal medium human preadipocyte growth medium (Cat No. 811-500;

113

Sigma-Aldrich). After reaching approximately 70% to 80% confluence, preadipocytes were

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transferred to 6-well plates (approximately 1.7 × 105 cells/well) and maintained in basal medium for

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48 h. After 48 h, cell differentiation was initiated with human adipocyte differentiation medium (Cat

116

No. 811D-250; Sigma-Aldrich). The differentiation medium was renewed every 3 days for 16 days.

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After cell differentiation, adipocytes were deprived of pre-existing hormones using human

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adipocyte starvation medium (Cat No. 811S-250; Sigma-Aldrich) for 24 h. After hormone

119

depletion, treatments were performed in triplicate for 24 h, and the supernatants and cells were

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collected for subsequent analyses. The control group (C) was not treated. The T3 group was treated

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with 10 nM T3 [33-36]. The irisin group (I) was treatment with 1 µg/ml (20 nM) irisin [17, 30].

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Cell viability assay

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The viability of human subcutaneous adipocytes was evaluated as described previously [37].

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T3 or irisin treated cells were incubated with 50 µl of 5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-

125

2,5-diphenyltetrazolium bromide) for 4 h at 37 oC. Then, the MTT-containing medium was aspirated

126

and 200 µl of dimethyl sulfoxide was added to lyse the cells and solubilize the water-insoluble

127

formazan. The absorbance of cell lysates at 540 nm was determined using a Synergy HT microplate

128

reader (Biotek Instruments, Winooski, VT, USA).

129 130 131

Differentiation of HPAd and evaluation of intracellular lipid accumulation following hormone

132

treatment

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After adipocyte differentiation and hormone treatment, the culture medium was removed and

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cells were washed twice with PBS. Following incubation with 1 ml of formaldehyde for 30 min at

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room temperature (approximately 27oC) , cells were washed three times with PBS and then stained

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with 300 µl of Oil Red O dye (Sigma-Aldrich) for 2 h at 37°C. Stained cells were washed three

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times with distilled water and placed in an oven to dry. Cells were then observed by phase contrast 4

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microscopy to evaluate adipocyte differentiation by Oil Red O staining of the lipid droplets. Fifteen

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random fields from each well were photographed at 10× magnification. The photographs were

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analyzed using Image J program (NIH, Bethesda, MD, USA) for quantification of lipid

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accumulation in the control and experimental conditions. In addition, adipocyte differentiation was

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evaluated using the AdipoRed assay kit (Lonza, Walkersville, MD, USA), according to the

143

manufacturer's instructions. Adipocytes were incubated for 10 minutes with the AdipoRed reagent

144

at room temperature. Plates were examined using excitation and emission wavelengths of 485 nm

145

and 572 nm, respectively, on the aforementioned Synergy HT plate reader.

146 147

TG and glycerol calorimetric assay

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After hormone treatments, the supernatant of each adipocyte cell culture was collected to

149

evaluate the release of TG by an enzymatic-colorimetric method using the BS-200 Chemistry

150

Analyzer (Mindray, Shenzhen, China), according to the manufacturer's protocol. In addition,

151

glycerol levels were evaluated using the Glycerol Colorimetric Assay Kit (Cayman Chemical

152

Company, Ann Arbor, MI), according to the manufacturer’s instructions, and the absorbance of

153

samples was quantified at 540 nm using the aforementioned Synergy HT plate reader.

154 155

DNA damage and lipid peroxidation assay

156

As an indicator of oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OHdG) levels were

157

evaluated using the DNA/RNA Oxidative Damage EIA kit (Cayman Chemical), according to the

158

manufacturer’s instructions. The culture supernatant of adipocytes grown under control and

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experimental conditions was collected. Quantification was performed by extrapolating the

160

absorbance of samples using a standard curve made using known concentrations of 8-OHdG. All

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analyses were performed on a Spectra Max 190 microplate reader (Molecular Devices, Sunnyvale,

162

CA, USA). In addition, lipid peroxidation was assayed by measuring the levels of malondialdehyde

163

(MDA) produced by the thiobarbituric acid (TBARS) reaction. Absorbance was measured at 540

164

nm. The concentration of MDA was expressed as µM of MDA/mg of protein [38].

165 166

Protein synthesis (western blotting)

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After treatment with T3 or irisin, adipocytes were washed twice with ice-cold PBS and lysed

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using 100 µl of lysis buffer containing 500 mM Tris (pH 8), 150 mM NaCl, 1% Triton X-100, 10%

169

sodium dodecyl sulfate (SDS), and 0.5% deoxycholate. The homogenate was centrifuged, the

170

supernatant was collected, and the total protein content was determined using the Bradford method

171

[39] with bovine serum albumin as the standard. Samples containing 25 µg of protein were

172

subjected to 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, 5

173

proteins were transferred to nitrocellulose membranes, which were then blocked for 1 h with 5%

174

fat-free milk at room temperature (approximately 27oC), and incubated overnight at 4oC with

175

primary antibodies at a dilution of 1:500 for mouse monoclonal anti-PPARγ (E-8: S7273; Santa

176

Cruz Biotechnology, Dallas, TX, USA), 1:5000 for rabbit polyclonal anti-leptin antibody (AB9749;

177

Sigma-Aldrich), 1:250 for mouse monoclonal anti-UCP-1 antibody (4E5: 293418; Santa Cruz

178

Biotechnology), 1:1000 for rabbit monoclonal anti-adiponectin antibody (C45B10, mAb #2789;

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Cell Signaling Technology, Danvers, MA, USA), and 1:10,000 for rabbit monoclonal anti-

180

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Sigma-Aldrich) and anti-FNDC5

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antibody (ab131390; Abcam). After washing with Tris-buffered saline containing Tween-20,

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membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at

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room temperature. Immune complexes were then detected using the ECL method, and the

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immunoreactive bands were analyzed using Gel Logic (6000 PRO) with Carestream MI software

185

and quantified using the GelPro program.

186 187

RNA extraction, preparation, and sequencing

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Total RNA was extracted from adipocytes using the TRIzol reagent (Invitrogen, Carlsbad,

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CA, USA) as previously described [33-36]. RNA samples were quantified in Qubit and the integrity

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of the RNA was analyzed using a Bioanalyzer 2100® (Agilent, Santa Clara, CA, USA). Three RNA

191

samples from each group were used to build each of the libraries. These libraries were constructed

192

using the TruSeq1 RNA Sample Prep kit v2 (Illumina, San Diego, CA, USA) following the protocol

193

suggested by the manufacturer. Runs were performed on the Illumina HiSeq2500 platform with 100

194

bp paired end reads. All laboratory procedures were carried out at the ESALQ Genomics Center

195

(Piracicaba, SP, Brazil).

196 197 198

Bioinformatic analysis Quality

control

of

the

obtained

reads

was

performed

through

FastQC.

199

(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Steps for adapter removal and low

200

quality read filtering were performed using Cutadapt and Trimmomatic software. The reads were

201

aligned and quantified in Kallisto (version 0.43.0) [40] using the Gh38 human genome index

202

(GRCh38.p12 release-92) (http://www.ensembl.org) as a reference. The abundance data (Counts)

203

generated were imported using the Bioconductor Tximport package from R [41] and used for the

204

analysis of differential gene expression with the Deseq2 package [42] (27). Deseq2 is based on the

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read count analysis method, generalized linear models, negative binomial distribution, and

206

shrinkage estimates for dispersions [42]. Multifactor design analysis was used to control the effect

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of biological variation between samples from the same condition. In cases of high variability, 6

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Deseq2 relies on genetic estimates to obtain results [42]. The abovementioned properties make

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Deseq2 a powerful tool for analysis of differential expression and biological variation [43].

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Differentially expressed (DE) genes were classified as UP and DOWN, considering FoldChange

211

(FC) values > 1.3 for the T and I groups, with a p value < 0.05. Gene Ontology (GO) enrichment

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analyses for biological processes (BP) were performed with the clusterProfiler package using the

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p.adjust false discovery rate and p < 0.05.

214 215

Statistical analyses

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All data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s

217

test, after performing the Kolmogorov Smirnov normality test. Data are expressed as mean ±

218

standard deviation. The significance level was set at 5%.

219 220

Results

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HPAd undergo classic adipocyte differentiation

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HPAd were grown under normal conditions and analyzed before differentiation occurred,

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while they had a pre-adipocyte phenotype (Fig. 1a). As expected, after 16 days of incubation in

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human adipocyte differentiation medium, the cells displayed the typical morphology of mature

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adipocytes, which was mainly characterized by the presence of several cytoplasmic lipid droplets

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(Fig. 1b). Oil Red O staining showed more evident lipid droplets, since lipids were stained red (Fig.

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1c).

228 229

HPAd maintain their viability after treatment with T3 or irisin

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Next, we examined whether treatment of cells with 10 nM T3 or 20 nM irisin for 24 h was

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cytotoxic. As expected, the MTT assay showed that both T3 and irisin treatment had no effect on

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cell viability compared to the control (absence of treatment) (Fig. 2).

233 234

T3 and irisin treatment leads to reduced lipid accumulation in human adipocytes

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To examine the effect of T3 and irisin on lipid accumulation in human adipocytes, treated

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cells were stained with Oil Red O (Fig. 3a-c) and AdipoRed (Fig. 3d). Compared with the control,

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T3 and irisin treatments reduced lipid droplet accumulation in human adipocytes by approximately

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25% and 20%, respectively (Fig. 3d)

239 240

Effects of T3 and irisin on the release of TG and glycerol from human adipocytes

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TG and glycerol levels in human adipocyte starvation medium were assessed after T3 or

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irisin treatment (Fig. 4). No significant difference was observed between TG levels of human 7

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adipocytes treated with T3 and those of the control (p > 0.05) (Fig. 4a). However, irisin treatment

244

significantly increased TG levels compared with the control (p < 0.001) (Fig. 4a). Also compared

245

with the control, glycerol levels were significantly reduced after T3 treatment (p < 0.05) and

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significantly increased after irisin treatment (p < 0.001) (Fig. 4b).

247 248

T3 and irisin reduce oxidative stress and DNA damage in human adipocytes

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Oxidative stress and DNA damage were assessed using the TBARS and 8-OHdG assays for

250

human adipocyte supernatants after T3 or irisin treatment (Fig. 5). Both T3 (p < 0.0001) and irisin

251

(p < 0.001) treatment significantly reduced TBARS-mediated production of MDA compared with

252

the control. In addition, significantly reductions in 8-OHdG levels were observed after treatment

253

with T3 (p < 0.001) and irisin (p < 0.001), compared with the control.

254 255

Effects of T3 and irisin on UCP1, leptin, adiponectin, PPARγ, and FNDC5 protein expression

256

in human adipocytes

257

UCP1, PPARγ, leptin, adiponectin, and FNDC5 protein expressions were evaluated in human

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adipocytes using immunoblotting analysis (Fig. 6). UCP1 protein expression was significantly

259

increased after T3 or irisin treatment compared with the control (both p < 0.05, Fig. 6a). Significant

260

increases of the levels of adiponectin (p < 0.001) and leptin (p < 0.05) were observed after T3

261

treatment compared with the control (Fig. 6b, c). On the contrary, the levels of PPARγ were

262

significantly diminished by T3 treatment (p < 0.001), but no significant change in PPARγ levels was

263

observed after irisin treatment (Fig. 6d). FNDC5 protein expression was significantly decreased

264

after T3 treatment compared with that in the control (p < 0.001) (Fig. 6e). Irisin treatment did not

265

significantly alter the production of FNDC5 compared with the control (Fig. 6e).

266 267

8

268

GO enrichment analyses for BP

269

Enriched GO terms within the molecular function domain are summarized in Tables 1, 2, 3,

270

and 4. In general, enriched terms were similar between the T3 and irisin treatment groups,

271

demonstrating increased mitochondrial function and polymerase II and decreased lipid storage.

272 273

Table 1 Gene ontology molecular function categories enriched in the set of genes up-regulated by

274

T3 GO

Description

Adjusted_p-

Number of genes Number of

Perc_enrichement

value

DE in GO

genes in GO

GO:0032543 Mitochondrial translation

1,17E-10

20

122

0,163934426

GO:0140053 Mitochondrial gene expression

9,83E-10

20

137

0,145985401

GO:0070125 Mitochondrial translational elongation

1,25E-09

15

86

0,174418605

GO:0070126 Mitochondrial translational termination

1,45E-08

15

88

0,170454545

GO:0033108 Mitochondrial respiratory chain complex

2,60E-03

8

8

0,091954023

2,56E-09

15

78

0,192307692

7,31E-08

16

113

0,14159292

1,41E-03

5

31

0,161290323

2,84E-03

3

11

0,272727273

assembly GO:0061418 Regulation of transcription from RNA polymerase II promoter in response to hypoxia GO:0043618 Regulation of transcription from RNA polymerase II promoter in response to stress GO:0051123 RNA polymerase II transcriptional preinitiation complex assembly GO:0045899 Positive regulation of RNA polymerase II transcriptional preinitiation complex assembly

275 276 277

Note: This table shows the GO terms (enriched in the set of genes up-regulated by T3) and their description, the adjusted p-value, the number (N) of DE genes contained in each of the GO definitions, the total number of genes present in the GO definitions, and the percent enrichment of DE genes within each definition.

278 279

Table 2 Gene ontology molecular function categories enriched in the set of genes down-regulated

280

by T3

GO

Description

Adjusted_p -value Number of genes

Number

DE in GO

Perc_enrichement

of genes in GO

GO:0019915 Lipid storage

3,56E-07

13

63

0,206349206

GO:0010876 Lipid localization

1,50E-06

34

380

0,089473684

GO:0010883 Regulation of lipid storage

1,19E-04

8

42

0,19047619

GO:0046486 Glycerolipid metabolic process

1,72E-03

29

441

0,065759637

5

17

0,294117647

GO:0010888 Negative regulation of lipid storage 2,75E-04

281 282 283

Note: This table shows: the GO terms (enriched in the set of genes down-regulated by T3) and their description, the adjusted p-value, the number (N) of DE genes contained in each of the GO definitions, the total number of genes present in the GO definitions, and the percent enrichment of DE genes within each definition.

284 285

Table 3 Gene ontology molecular function categories enriched in the set of genes up-regulated by 9

286

irisin

GO

Description

Adjusted_p-value Number of genes Number of Perc_enrichement DE in GO

genes in GO

GO:0032543 Mitochondrial translation

3,36E-32

55

122

0,450819672

GO:0070125 Mitochondrial translational elongation

2,44E-31

46

86

0,534883721

GO:0140053 Mitochondrial gene expression

4,86E-31

57

137

0,416058394

GO:0070126 Mitochondrial translational termination

1,46E-29

45

88

0,511363636

GO:0033108 Mitochondrial respiratory chain complex assembly

2,10E-04

16

87

0,183908046

GO:0043618 Regulation of transcription from RNA polymerase II

7,48E-15

35

113

0,309734513

5,68E-14

28

78

0,358974359

GO:0006369 Termination of RNA polymerase II transcription

2,35E-06

17

69

0,246376812

GO:0006368 Transcription elongation from RNA polymerase II

1,96E-03

16

106

0,150943396

2,36E-03

12

70

0,171428571

promoter in response to stress GO:0061418 Regulation of transcription from RNA polymerase II promoter in response to hypoxia

promoter GO:0042795 snRNA transcription from RNA polymerase II promoter

287 288 289

Note: This table shows: the GO terms (enriched in the set of genes down-regulated by irisin) and their description, the adjusted p-value, the number (N) of DE genes contained in each of the GO definitions, the total number of genes present in the GO definitions, and the percent enrichment of DE genes within each definition.

290 291

Table 4 Gene ontology molecular function categories enriched in the set of genes down-regulated

292

by irisin

GO

Description

Adjusted_p-value

Number of genes Number of genes Perc_enrichement DE in GO

in GO

GO:0019915

Lipid storage

2,12E-07

19

63

0,301587302

GO:0010876

Lipid localization

7,61E-08

61

380

0,160526316

GO:0010883

Regulation of lipid storage

1,28E-05

13

42

0,30952381

GO:0046486

Glycerolipid metabolic process

3,29E-07

66

441

0,149659864

293 294 295

Note: This table shows: the GO terms (enriched in the set of genes down-regulated by irisin) and their description, the adjusted p-value, the number (N) of DE genes contained in each of the GO definitions, the total number of genes present in the GO definitions, and the percent enrichment of DE genes within each definition.

296 297

Discussion

298

Our study provides evidence that treatments with T3 and irisin are able to modulate the

299

thermogenic pathways, leading to the browning of human white adipocytes; increase the protein

300

levels of UCP1, one of the most important markers of this process; and decrease lipid droplet

301

accumulation. The reduction in oxidative stress and DNA damage observed after T3 and irisin

302

treatments suggests proper cell and mitochondrial function to support heat production. We observed

303

high adiponectin and leptin levels following treatment with T3, but irisin maintained the basal 10

304

levels of these adipokines. In addition, modulation of lipolysis could be associated with the high TG

305

and glycerol levels after irisin treatment.

306

Irisin is an exercise-induced myokine. Recent evidence indicated an important metabolic

307

function of irisin in AT [17]. In the current study, both T3 and irisin decreased intracellular lipid

308

accumulation (Fig. 3a, d). Lipolysis involves the degradation of TG and the release of glycerol,

309

among other molecules [44]. T3 treatment was able to maintain TG and decrease glycerol levels,

310

whereas irisin elevated TG and glycerol levels (Fig. 4a, b).

311

Human WAT deposits can differentiate into brown adipocytes and acquire a greater capacity

312

to oxidize intra-adipose fatty acids [45]. This could explain the decrease in lipid accumulation after

313

T3 treatment and the absence of an effect on TG or extracellular glycerol levels. Gao et al. [46]

314

detected increased glycerol levels in 3T3-L1 adipocytes treated with human recombinant irisin,

315

which suggested that irisin has important roles in glucose and lipid metabolism by promoting lipid

316

degradation. Our data are consistent with this suggestion.

317

Abnormal lipid accumulation can lead to oxidative stress by increasing the production of

318

reactive oxygen species (ROS) [47] that consequently damage lipids, proteins, and DNA [48]. We

319

evaluated the effect of T3 and irisin on the levels of MDA to assess lipid peroxidation, a mechanism

320

through which ROS attack fatty acids and 8-OHdG, and, in turn, cause DNA damage. DNA damage

321

may occur at the level of nitrogenous bases [49]. Guanine is more susceptible to oxidation. When

322

oxidized, guanine incorporates a hydroxyl group at the eighth carbon of the molecule, forming 8-

323

OH-dG [50]. Presently, a decrease in MDA and 8-OHdG levels in the presence of T3 and I was

324

observed (Fig. 5a, b). De Sibio et al. [8] demonstrated that weight loss decreased DNA damage in

325

obese rats subjected to caloric restriction. The observation corroborates our results, since both T3

326

and I treatments produced a reduction in intracellular lipid accumulation, suggesting an improved

327

cellular environment that is able to properly control physiological processes.

328

The stimulation of browning may promote body fat reduction [51, 49]. Several

329

mechanisms have been proposed for WAT browning [53], including prolonged cold exposure,

330

increased UCP1 expression, and use of thermogenic hormones. We observed that treatment of

331

human subcutaneous adipocytes with T3 or irisin increased UCP1 protein expression (Fig. 6a).

332

Previously, exogenous irisin led to the browning of fat by increasing UCP1 mRNA and protein

333

levels, resulting in a significant reduction in body weight and an improvement in glucose tolerance

334

in mice and rats [17]. In agreement with the results reported by Skarulis et al. [54], we observed a

335

T3-mediated increase in UCP-1 levels and reduction in lipid accumulation. Raschke et al. [55]

336

suggested that the beneficial effect of irisin observed in mice is unlikely to be translated to humans.

337

In contrast to our study, in which mature adipocytes were incubated with irisin, the incubation in the

338

prior study involved preadipocytes, which did not differentiate to "Brite" (brown-in-white) human 11

339

adipocytes when incubated with recombinant FNDC5 or irisin, or display changes in the expression

340

of UCP1 and PPARγ mRNAs. In contrast to Raschke et al. [55], Kristóf et al. [56] found that irisin

341

treatment during white adipocyte differentiation up-regulated the expressions of UCP1 and PPARγ,

342

among other proteins, proving the effectiveness of irisin for body weight reduction.

343

Presently, the expression of FNDC5 was suppressed in human subcutaneous adipocytes in

344

the presence of T3. This was correlated with the increased levels of adiponectin and leptin, and with

345

the maintenance of PPARγ protein expression. These events may be directly related, since FNDC5

346

synthesis is stimulated by PPARγ [52,57]. Our results are in agreement with the findings of Pérez-

347

Sotelo et al. [57], who showed that inhibition of FNDC5 in the AT of obese patients resulted in

348

increased adiponectin production and maintenance of PPARγ levels. However, in contrast to our

349

results, UCP1 production was previously reported to decrease. This discrepancy is probably due to

350

the use of T3 in our study, which stimulated UCP1 protein expression and had a direct effect on

351

thermogenesis [58]. Dittner et al. [59] demonstrated that thyroxine elevated the levels of UCP1

352

protein expression in mouse brown adipocytes, but had no effect on its expression in WAT. The

353

presence of exogenous irisin maintained the production of adiponectin, leptin, PPARγ, and FNDC5

354

(Fig. 6b, e).

355

GO analysis demonstrated that T3 and irisin (Tables 2 and 4, respectively) down-regulated

356

genes involved in lipid storage, such as lipoprotein lipase (LPL) (data not shown), a process

357

contrary to obesity in which AT stores lipids and causes insulin resistance. LPL is important in the

358

regulation of the release of free fatty acids from TG-rich lipoproteins [60]. Decreased LPL activity

359

in AT causes loss of fatty acid uptake by lipoproteins and increases lipolysis [61]. This may be

360

consistent with previous data regarding glycerol and TG levels and decreased lipid accumulation.

361

Tables 1 and 3 show that treatment with T3 and irisin resulted in up-regulation of mitochondrial

362

activity. THs regulate lipid metabolism, including lipogenesis, lipolysis, and thermogenesis. Our

363

results are in agreement with the findings of Lee et al. [62], who demonstrated that T3 treatment

364

increased the oxygen consumption rate, suggesting that T3 induces energy utilization in white

365

adipocytes by regulating UCP-1 expression and mitochondrial biogenesis. An enrichment of BP

366

terms related to mitochondrial activity was also observed in the I group. In addition, we showed that

367

T3 and irisin increased gene transcription, as the BP for RNA polymerase II was enriched within

368

the up-regulated genes, which have a major impact on cell differentiation and maintenance.

369

Irisin has been proposed to regulate energy homeostasis [63]. Our results implicate irisin

370

with metabolic parameters, especially those influencing pathways involved in lipid metabolism. We

371

observed a decrease in lipid accumulation that probably reflected increased lipolysis, unaltered

372

levels of adipokines associated with appetite control (leptin and adiponectin), and maintenance of

373

insulin sensitivity. The maintenance of PPARγ has a well-established role in driving 12

374

adipogenesis/TG storage and fatty acid oxidation in fat cells [64].

375

In the present study, we explored the impact of T3 and irisin in directing the browning of

376

human adipose subcutaneous cells and the different destinations for the products generated

377

following a decrease in intracellular lipid accumulation, increase in UCP1 protein expression

378

(browning marker), and modulation of PPARγ, FNDC5, and adipokines by T3. The data indicate

379

that irisin improves the lipid profile, oxidative stress, and DNA damage, while maintaining the

380

levels of leptin and adiponectin. These effects could be used for the prevention of obesity.

381

Furthermore, the use of analogues of T3 could stimulate leptin production in cases of its deficiency

382

and elevate adiponectin levels in cases of obesity to improve insulin sensitivity.

383 384

Acknowledgments

385 386

Funding from FAPESP (2016/03242-3) and CAPES (409438/2016) supported this study. The

387

funders had no role in the study design, data collection and analysis, decision to publish, or

388

preparation of the manuscript. This manuscript has been proofread by native English speakers, the

389

edit was performed by professional editors at Editage, a division of Cactus Communications (JOB

390

CODE CENOG_3).

391 392

Disclosure Summary:

393

The authors have nothing to disclose.

394 395

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Figure legends

626

Fig. 1. HPAd before and after differentiation into adipocytes. a) Non-differentiated HPAd. b)

627

HPAd after 10 days of differentiation. c) HPAd stained with Oil Red O after 10 days of

628

differentiation. Arrows show adipocytes with cytoplasmic lipid droplets.

629 630

Fig. 2. Adipocyte viability. After 24 h of treatment with 10 nM T3 or 20 nM irisin, cells were

631

incubated with 50 µl of MTT reagent for 4 h. Absorbances were measured at 540 nm. T= 0 nM T3,

632

I = 20 nM irisin. Data are expressed as mean ± standard deviation (ANOVA supplemented with

633

Tukey's test); n = 3, ns: non-significant. MTT: Methylthiazolyldiphenyl-tetrazolium bromide, T:

634

Triiodothyronine, I: Irisin.

635 636

Fig. 3. T3 and irisin alters lipid accumulation in subcutaneous adipocytes. (a-c)

637

Photomicrography of Oil Red O stained adipocytes after T3 or irisin treatment, (d) Decreased lipid

638

accumulation in adipocytes shown by AdipoRed quantification. T = 10 nM T3, I = 20 nM irisin. All

639

data are expressed as mean ± standard deviation. (ANOVA supplemented with Tukey's test); n = 3,

640

*** p < 0.001 indicated a statistically significant difference. T: Triiodothyronine, I: Irisin.

641 642

Fig. 4. Effect of T3 and irisin on triglyceride and glycerol release. a) Triglyceride (TG) levels

643

were measured using an enzymatic-colorimetric method after treatment with T3 or irisin for 24 h. b)

644

Release of glycerol after T3 or irisin treatment for 24 h. T = 10 nM T3, I = 20 nM irisin. Data are

645

expressed as mean ± standard deviation (ANOVA supplemented with Tukey's test); n = 3. * p <

646

0.05, *** p < 0.001, ns: nonsignificant. T: Triiodothyronine, I: Irisin.

647 648

Figure 5. Malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) concentrations

649

in cell culture medium after treatment with T3 or irisin. a) Quantification of 8-OH-dG by

650

ELISA to assess DNA damage, b) MDA concentration was assessed by the TBARS assay and was

651

normalized to the total amount of protein. T = 10 nM T3, I = 20 nM irisin, TBARS = thiobarbituric

652

acid reactive substance. Data are expressed as mean ± standard deviation (ANOVA supplemented 20

653

with Tukey's test); n = 3. *** p < 0.001. T: triiodothyronine, I: irisin.

654 655

Figure 6. T3 and irisin alter protein synthesis in subcutaneous adipocytes. (a) Influence of T3

656

and irisin on UCP1 protein levels. (b) Influence of T3 and irisin on adiponectin protein expression.

657

(c) Influence of T3 and irisin on leptin protein expression (d) Influence of T3 and irisin on PPARγ

658

protein expression. (e) Influence of T3 and irisin on FNDC5 protein expression. (f) Representative

659

images of immunoblots are shown, equal amounts of solubilized proteins were immunoblotted with

660

antibodies against UCP1, adiponectin, leptin, PPARγ, FNDC5, or GAPDH. Quantitative analysis of

661

immunoblot images was performed using Gel Logic (6000 PRO) with Carestream MI software

662

integrated optical density. T = 10 nM T3, I = 20 nM irisin. Data are expressed as mean ± standard

663

deviation (ANOVA supplemented with Tukey's test); n = 3. * p < 0.05, *** p < 0.001, ns: non-

664

significant. T: Triiodothyronine, I: irisin.

665 666

Graphical Abstract. T3 and irisin regulate energy homeostasis. Effects of irisin are represented

667

with red arrows, while those of T3 are represented with black arrows. Irisin induced a decrease in

668

lipid accumulation through lipolysis, improved oxidative stress and DNA damage, maintained the

669

levels of adipokines that are associated with appetite control and insulin sensitivity (leptin and

670

adiponectin), and maintained PPARy levels. T3 induced a decrease in lipid accumulation through

671

the oxidation of fatty acids in adipocytes; improved oxidative stress and DNA damage; increased

672

the production of adipokines, adiponectin, and leptin; maintained PPARy levels; and decreased the

673

production of FNDC5.

21

a

b



Figure 1.

c

Figure 2.

a

c

b



500µM

d

Figure 3.



500µM



500µM

a

Figure 4.

b

a

b

Figure 5.

c

b

a

e

d

C

T

I

f UCP-1

Figure 6.

33kDa

Adiponectin

27kDa

Leptin

16kDa

PPARy

50kDa

FNDC5

22kDa

GAPDH

37kDa

Irisin Effects

T3 Effects

↑ increase ↓ decrease *unchanged

↑ increase ↓ decrease

protein synthesis

↑↑ UCP1 ↓↓ Intracellular lipid accumulation

T3

Cytoplasm

Irisin

Lipidic Droplet

↑ ↔ Triglicerides ↑ ↓ Glycerol

↓↓ Oxidative stress (Malonaldehyde-MDA)

↓↓ DNA Damage

mRNA

Nucleus

* ↓ PPARγγ ↑ Leptin ↑ Adiponectin ↓ FNDC5 Graphical Abstract

Highlights:

1. T3 and Irisin diminished intracellular lipid accumulation and cellular damage 2. Upregulation of UCP1 in adipocytes by T3 and irisin 3. Upregulation of adiponectin and leptin by T3 4. Maintenance of adiponectin and leptin levels by irisin 5. FNDC5 synthesis in human subcutaneous adipocytes 6. T3 decreased FNDC5 and PPARy