MicroRNA miR-222 mediates pioglitazone beneficial effects on skeletal muscle of diet-induced obese mice

Journal Pre-proof MicroRNA miR-222 mediates pioglitazone beneficial effects on skeletal muscle of diet-induced obese mice Mariana de Mendonça, Érica de Sousa, Ailma O. da Paixão, Bruna Araújo dos Santos, Alexandre Roveratti Spagnol, Gilson M. Murata, Hygor N. Araújo, Tanes Imamura de Lima, Dimitrius Santiago Passos Simões Fróes Guimarães, Leonardo R. Silveira, Alice C. Rodrigues PII:

S0303-7207(19)30363-6

DOI:

https://doi.org/10.1016/j.mce.2019.110661

Reference:

MCE 110661

To appear in:

Molecular and Cellular Endocrinology

Received Date: 2 August 2019 Revised Date:

19 November 2019

Accepted Date: 19 November 2019

Please cite this article as: de Mendonça, M., de Sousa, É., da Paixão, A.O., Araújo dos Santos, B., Spagnol, A.R., Murata, G.M., Araújo, H.N., Imamura de Lima, T., Passos Simões Fróes Guimarães, D.S., Silveira, L.R., Rodrigues, A.C., MicroRNA miR-222 mediates pioglitazone beneficial effects on skeletal muscle of diet-induced obese mice, Molecular and Cellular Endocrinology (2019), doi: https:// doi.org/10.1016/j.mce.2019.110661. 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. © 2019 Published by Elsevier B.V.

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MicroRNA miR-222 mediates pioglitazone beneficial effects on skeletal muscle of diet-

2

induced obese mice

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Mariana de Mendonça1, Érica de Sousa1, Ailma O. da Paixão1, Bruna Araújo dos Santos1,

5

Alexandre Roveratti Spagnol1, Gilson M. Murata2, Hygor N. Araújo3, Tanes Imamura de

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Lima3, Dimitrius Santiago Passos Simões Fróes Guimarães3, Leonardo R. Silveira3, Alice

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C. Rodrigues*1.

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1

Department of Pharmacology and 2Department of Physiology and Biophysics, Institute of

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Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil; 3Obesity and

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Comorbidities Research Center, Campinas, Sao Paulo, Brazil; 4Department of Structural

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and Functional Biology, Institute of Biology, University of Campinas (UNICAMP),

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Campinas, Sao Paulo, Brazil

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Correspondence:

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Alice Cristina Rodrigues, PhD

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[email protected]

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Keywords: MicroRNA, obesity, insulin resistance, Ppargamma

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Abstract

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Pioglitazone belongs to the class of drugs thiazolidinediones (TZDs) and is an oral

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hypoglycemic drug, used in the treatment of type 2 diabetes, which improves insulin

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sensitivity in target tissues. Adipose tissue is the main target of pioglitazone, a PPARg and

31

PPARa agonist; however, studies also point to skeletal muscle as a target. Non-PPAR

32

targets of TZDs have been described, thus we aimed to study the direct effects of

33

pioglitazone on skeletal muscle and the possible role of microRNAs as targets of this drug.

34

Pioglitazone treatment of obese mice increased insulin-mediated glucose transport as a

35

result of increased fatty acid oxidation and mitochondrial activity. PPARg blockage by

36

treatment with GW9662 nullified pioglitazone’s effect on systemic and muscle insulin

37

sensitivity and citrate synthase activity of obese mice. After eight weeks of high-fat diet,

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miR-221-3p expression in soleus muscle was similar among the groups and miR-23b-3p

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and miR-222-3p were up-regulated in obese mice compared to the control group, and

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treatment with pioglitazone was able to reverse this condition. In vitro studies in C2C12

41

cells suggest that inhibition of miR-222-3p protects C2C12 cells from insulin resistance

42

and increased non-mitochondrial respiration induced by palmitate. Together, these data

43

demonstrate a role of pioglitazone in the downregulation of microRNAs that is not

44

dependent on PPARg. Moreover, miR-222 may be a novel PPARg-independent

45

mechanism through which pioglitazone improves insulin sensitivity in skeletal muscle.

46 47 48 49 50

3 51 52

Introduction

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Obesity is associated with metabolic and immunological dysfunctions, including insulin

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resistance, that can result on the development of type 2 diabetes mellitus (Lee & Lee,

55

2014; Ye, 2013). Pioglitazone is a PPARalpha (PPARa) and mainly PPARgamma

56

(PPARg) ligand; both are receptors that control the expression of genes involved in lipid

57

metabolism. Pioglitazone is an insulin sensitizer used as an oral hypoglycemic to treat type

58

2 diabetes, and it is within the class of drugs thiazolidinediones (TZDs) (Lehmann et al.,

59

1995).

60

Adipose tissue is the main target of PPARg agonists; however, studies also point to

61

skeletal muscle as a target (Zierath et al., 1998; Hallakou et al., 1998; Rabol et al., 2010).

62

The beneficial effects of TZD drugs have been attributed to adiponectin action (Kubota et

63

al., 2006; Maeda et al., 2001; Mendonça et al., 2019), a hormone known to be secreted

64

mainly by adipocytes. However, pioglitazone-induced improvement of insulin resistance

65

and diabetes is mediated via adiponectin-independent pathways in skeletal muscle (Kubota

66

et al., 2006; Mendonça et al, 2019).

67

Recently, we have shown pioglitazone induces adiponectin receptor 2 (AdipoR2) signaling

68

in the absence of adiponectin (Mendonça et al., 2019), a phenomenon that seems to be

69

mediated by microRNA miR-150-5p that targets Adipor2 3’UTR (Li et al., 2016).

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Adiponectin receptors (AdipoR1 and AdipoR2) activation in the muscle has been

71

associated with increased glucose uptake and fatty acid oxidation (Tomas et al., 2002;

72

Yamauchi et al., 2002; Li et al 2007). Additionally, AdipoR2 activity induces PPARa

73

expression, which is involved in fatty acid metabolism (Yamauchi et al 2007).

74

MicroRNAs (miRs) are a class of non-coding RNAs that have around 19-22 nucleotides

75

(Ambros, 2004). They can be encoded in intergenic or intron regions of other genes and

4 76

are generally involved in the post-transcriptional regulation of gene expression (Bartel,

77

2004). Several studies have already demonstrated that microRNAs are important

78

mediators of several comorbidities associated with obesity (Zaiou, El Amri, & Bakillah,

79

2018), showing the potential of these molecules as a possible treatment.

80

MicroRNAs have been shown to be regulated by PPARs nuclear receptors (Portius,

81

Sobolewski, & Foti, 2017). Of note, pioglitazone and rosiglitazone have been shown to

82

modulate the expression of 27 different miRNAs in human subcutaneous and visceral

83

adipocytes (Dharap, Pokrzywa, Murali, Kaimal, & Vemuganti, 2015; J. Yu et al., 2014).

84

Specifically, the expression of microRNAs miR-221 and miR-222 has a positive

85

correlation with obesity (Chartoumpekis et al., 2012; Meerson et al., 2013), and miR-23b

86

expression is increased in soleus muscle of obese mice (Frias et al., 2018).

87

In this study, we hypothesized that pioglitazone may regulate microRNAs miR-221/222

88

and miR-23b in order to promote its beneficial actions on soleus muscle, such as

89

improvements in insulin resistance.

90 91

Research Design and Methods

92 93

Animals

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Male wild-type C57BL/6J mice were obtained from the Facility for Mice Production at the

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Department of Pharmacology of the Institute of Biomedical Sciences (ICB) of the

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University of Sao Paulo (USP), and were maintained at 12:12-h light–dark cycle and

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23°C±2°C. The animals were housed in cages (2-3 animals/cage) and received standard

98

diet (Nuvilab-Nuvital Nutrients Ltd., Parana, Brazil) and water ad libitum until to the

99

beginning of the experimental period. The Experimental Animal Ethics Committees of

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ICB-USP approved the experimental procedure of this study (Protocols Numbers:

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165/2011 and 137/2015).

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Experimental Design

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Thirty male wild-type C57BL/6J mice, with 8 weeks of age were randomly divided into

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two groups: balanced diet (C group) (cod151: 9% fat, 15% protein and 76% carbohydrate

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of total kcal), and high-fat diet (H group) (cod10: 59% fat, 15% protein and 26%

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carbohydrate, as a percentage of total kcal) (Pragsoluções Biociências, Jaú, Sp, Brazil).

108

After 6 weeks of diet, hydrochloride pioglitazone (EMS S/A, São Bernardo do Campo, SP,

109

Brazil), was added to the diet (35 mg/kg of b.w./day) of a portion of the animals in group

110

H. These mice were treated for the last 2 weeks of the protocol, creating one new group:

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high-fat diet plus pioglitazone (HP). Intake of pioglitazone was controlled by ensuring

112

mice ate all of the provided diet each day. Body weight was measured every week and

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food intake every 3 days. By the end of 8 weeks, animals were euthanized (between 1 -3

114

pm) by decapitation and soleus and gastrocnemius muscles were carefully dissected from

115

the surrounding tissue, frozen in liquid nitrogen and stored at -80 ̊C. For histochemical

116

analysis, soleus muscles were immediately frozen in liquid nitrogen-cooled isopentane.

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Retroperitoneal, mesenteric and epididymal adipose tissue depots were dissected and

118

weighed to evaluate adiposity level, and blood was collected for metabolite determinations

119

in serum.

120

To investigate the mechanism by which pioglitazone increases insulin sensitivity in diet-

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induced obese mice, fifty male C57BL/6J mice were subjected to the same experimental

122

protocol described above were divided into additional two groups: one fed with high-fat

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diet (HFD) and treated with pioglitazone plus PPARa antagonist GW6471 (Cayman

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Chemical CAS 880635-03-0) 10mg/Kg of b.w. (HP+GW6471 group) (Xu et al., 2002),

125

and the other fed with HFD and treated with pioglitazone plus the PPARg antagonist

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GW9662 (Cayman Chemical CAS 22978-25-2) 10mg/Kg of b.w. (HP+GW9662 group)

127

(Nakano et al., 2006). At the 11th day of treatment, an insulin tolerance test was performed

128

and by the end of 8 weeks, animals were euthanized by decapitation, blood was collected

129

and soleus muscle was carefully dissected from the surrounding tissue, frozen in liquid

130

nitrogen and stored at -80 ̊C.

131

The number of individual experiments was representative of at least two litters. The total

132

number of animals used in each experiment is indicated in the figure legends.

133 134

Insulin tolerance test (ITT) and Serum metabolite measurements

135

For ITT all groups were fasted for 6 hours and blood was collected from the tail vein at 0,

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4, 8, 12, 16 and 20min after intraperitoneal insulin injection (0.75 mUI per g b.w.) (Bonora

137

et al., 1989). Blood glucose level was measured using a glucometer (Accu-Chek, Roche,

138

USA). To estimate insulin sensitivity, blood glucose disappearance rate (KITT) was

139

calculated during the 4 to 20 min period.

140

After euthanasia, insulin levels from serum were measured by ELISA using Rat/Mouse

141

Insulin ELISA kit (EZRMI-13K) (EMD Millipore, Billerica, MA, USA). Total

142

Cholesterol (K083-2), LDL (K088), HDL (K015), and TG (K117-1) concentrations were

143

determined using specific kits (Bioclin, Belo Horizonte, BH, Brazil).

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Total RNA Isolation

146

Total RNA was extracted from mouse soleus muscle using TRIzol reagent (Thermo Fisher

147

Scientific), according to the manufacturer's instructions. The concentration and purity of

148

total RNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher

149

Scientific).

150

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Quantification of microRNA expression

152

The relative quantification of miRNAs miR-23b-3p, miR-221-3p and miR-222-3p was

153

performed by real-time PCR using the miRCURY LNA ™ microRNA system (Exiqon).

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Universal cDNA synthesis was performed from 10ng of total RNA obtained from the

155

soleus muscle, using the Universal cDNA synthesis kit (Exiqon). The reagents were mixed

156

and incubated in a thermocycler for 60 min at 42 °C and 5 min at 95 °C. The cDNA

157

obtained was diluted 40x in nuclease-free water and stored at -20 °C. The primers required

158

for each miRNA were purchased from Exiqon. The expression of the miRNAs was

159

performed by qPCR with detection by SYBR Green, using the ABI Prism 7500 (Life

160

Technologies), and following the universal amplification protocol: 95°C for 10 min

161

followed by 40 cycles of 95°C for 15s and 60°C for 1 min, followed by a dissociation

162

curve. For the quantification of gene expression, the relative quantification method was

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used using constitutive gene SNORD110 as reference using the 2-∆∆CT method (Livak &

164

Schmittgen, 2001). SNORD110 was chosen as a reference gene as it expression was not

165

affected by diet or treatment.

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Quantification of mRNA expression

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For mRNA expression, cDNA was synthetized from 500ng (mouse soleus muscle) of total

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RNA extract using High Capacity cDNA Reverse Transcription Kit (Thermo Scientific).

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All PCR reactions were performed using a diluted (1/10) cDNA template, forward and

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reverse primers (200 nM each) and Power SYBR Green PCRMaster Mix (Thermo-

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Fisher).For mRNA expression, the genes analyzed included: Ppara, Pparg, AdipoQ,

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AdipoR1, AdipoR2,Cebpa, Cpt1b, Slc2a4, Pgc1α, Ucp3, Rplp0 and Hprt1 (Table 1). RT-

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qPCR was performed using an ABI Prism 7500 (Thermo Fisher Scientific) following the

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universal amplification protocol: 95°C for 10 min followed by 40 cycles of 95°C for 15s

8 176

and 60°C for 1 min, followed by a dissociation curve. For the quantification of gene

177

expression, the relative quantification method was used, using constitutive genes Rplp0

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and Hptr1 as reference using the 2-∆∆CT method (Livak & Schmittgen, 2001). All

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primers were tested for amplification efficiency by qPCR which was around 100% for all

180

targets.

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Enzyme activity assays

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Gastrocnemius and soleus muscles were ground to a powder in liquid nitrogen and 20 mg

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was homogenized with 1:100 (wt/vol) of extraction buffer pH7,4 (50mM Tris-HCl, 1mM

185

EDTA and protease inhibitor (Sigma P8340)). Samples were centrifuged for 10min at

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12000rpm at 4oC, and the supernatant was used for determination of exyme activity.

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Determinations

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dehydrogenase activity were determined as previously described (Alp, Newsholme, &

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Zammit, 1976; Crabtree, Higgins, & Newsholme, 1972; Lynen, 1955). For β-hydroxyacyl

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CoA dehydrogenase activity we used acetoacetly coA as a substrate. The samples were

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read in a Spectramax M5 spectrophotometer (Molecular devices, CA, USA). The results

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were expressed on a protein basis as determined by the BCA protein assay kit (Thermo

193

Fisher Scientific). Enzyme activities were assessed in triplicate and measurements were

194

performed every 10 seconds over a 3 min period.

of pyruvate kinase,

citrate

synthase

and

β-hydroxyacyl

CoA

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Histochemical analyses

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Frozen muscles were cut in 10 µm-thick sections from the medium portion of soleus or

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gastrocnemius using a cryostat Leica CM 3050S and used for succinate dehydrogenase

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(SDH) evaluation. SDH activity was estimated by reduction of nitroblue tetrazolium

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(NBT) using 1-methoxyphenazine methosulfate (mPMS) (Blanco, Sieck, & Edgerton,

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1988). Images were acquired using a bright field Nikon E1000 microscope (Melville, NY,

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USA). Images of soleus and gastrocnemius muscles at 200X magnification were captured

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using a Nikon DMX1200 digital camera. For each muscle, 2-3 non-overlapping regions

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were imaged and at least 100 fibers were analyzed per each animal and quantified using

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Image J analysis software (Image J software, NIH, USA). Two observers (ES and ARS),

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with no knowledge of the groups studied, performed the results of stain analysis.

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Western Blot

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The two soleus muscles from all groups were isolated and incubated as previously

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described, with minor modifications (Frias Fde et al., 2016). One intact soleus muscles

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from each animal was briefly incubated and agitated for 20 minutes at 35°C in Krebs–

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Ringer bicarbonate buffer containing 5.6 mM glucose, pH 7.4, (pre-gassed for 30 min with

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95% O2/5% CO2). The other soleus muscle was incubated in exactly the same conditions

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but with 7 nM of insulin. Next, each soleus muscle was homogenized in a cell disruption

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buffer from miRvana PARIS kit (Thermo Fisher Scientific, Waltham, MA, USA), and

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protein concentration was determined by the Bradford method. Thirty micrograms of

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protein were boiled, fractionated in 10% SDS-PAGE and transferred onto a nitrocellulose

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membrane. Membranes were blocked in 5% non-fat milk diluted in TBS-Tween for 1 hour

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at room temperature. Membranes were incubated overnight at 4oC with primary antibodies

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(p-AKT (Ser473) Cell Signalling #9018, Gapd, Abcam ab181602). Detection was

221

performed by C-Digit Imager (LI-COR, Lincoln, NE, USA) after incubation with

222

peroxidase-conjugated secondary antibodies for 2h at room temperature, using the

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“ClarityTM Western ECL Substrate” detection system (Bio-Rad, Hercules, CA, EUA).

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Protein expression was normalized with ponceau staining for soleus muscle (Fortes et al.,

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2016).

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Cell culture conditions and treatments

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The C2C12 cell line (ATCC #: CRL-1772) was used as the mouse skeletal muscle model.

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C2C12 cells were maintained in DMEM (SIGMA, St. Louis, MO) with 10% fetal bovine

230

serum and 1% penicillin/streptomycin (10,000 UI/mL streptomycin and 10,000 UI/mL

231

penicillin) under humidified condition with 5% CO2 at 37ºC. The culture medium was

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changed three times per week. For differentiation of myocytes into myotubes, DMEM

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containing horse serum (2%).

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Treatment of C2C12 cells

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Anti-miRs (Exiqon) from miR-23b and miR-222 were obtained and were transfected in 3-

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days differentiated C2C12 cells. Transfections were performed using Jetprime®

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(Polyplus). 50,000 cells were seeded in 12-well plates, and after reaching 100%

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confluency were differentiated into myotubes for 5 days. The myotubes were transfected

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with 1 µL mixture of Jetprime and 25 pmol/µL of the inhibitor in 100 µl of buffer added to

241

1mL of media. After 48h, the efficiency of the transfection was evaluated by quantifying

242

the expression of microRNAs by real-time PCR. As a negative control, a random sequence

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was used that did not affect the expression of the microRNAs. After verification of

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transfection efficiency, myotubes transfected with miR-23b or miR-222 inhibitors or

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control were treated with 0.75 mM of palmitic acid for 16h and stimulated with 100 nM

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insulin for 15 min. Insulin signaling, indicated by the phosphorylation of AKT (ser 473),

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was evaluated by western blot as described above. Mitochondrial oxygen consumption

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was also evaluated, as previously described (Lima et al., 2019) using a Seahorse analysis

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(XF24; Agilent Technologies Inc., Santa Clara, CA, USA). Non-mitochondrial OCR

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values were subtracted from all data before being used for the analyses. All Seahorse

11 251

measurements were normalized by protein quantified by the Bradford assay.

252

To verify the direct effect of pioglitazone in muscle, differentiated C2C12 cells were

253

treated with vehicle (control) or 0.75mM of palmitic acid or palmitic acid + 50 µM

254

pioglitazone for 24 hours. After treatment, the cells were washed twice with cold PBS and

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total RNA was isolated using TRIzol reagent (Thermo Scientific) for analysis of miR-23

256

and miR-222 expression.

257 258

C2C12 cells stably expressing PGC-1α

259

PGC1α 2.4 kb sequence was extracted from pCDNA-PGC-1α plasmid (Origene) and

260

inserted in a pBABE empty vector resulting in pBABE-PGC-1α plasmid. This

261

construction was transfected in HEK293T cells for production of retroviruses. C2C12

262

myoblasts at 40-50% confluence were transduced with pBABE-empty or pBABE-PGC1α

263

viral particles and positive clones were selected by adding puromycin (1μg/mL) after 24

264

hours of transduction. After 4-6 days of selection, cells were assayed for PGC-1α mRNA

265

and protein levels. Cells overexpressing Pgc1-alpha and its control were washed twice

266

with cold PBS and total RNA was isolated using TRIzol reagent (Thermo Scientific) for

267

analysis of miR-23b and miR-222 expression.

268 269

Statistical analyses

270

The results are presented as mean ± S.E.M. and were analyzed by either Student “t” test,

271

or one-way ANOVA followed by Tukey post-test. The significance level was set at

272

p<0.05.

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Results

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Pioglitazone treatment reverts insulin resistance promoted by high-fat diet

12 276

After eight weeks of diet, mice fed a high-fat diet (H) gained more weight than mice fed a

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balanced diet (C) (Table 2). Regarding the composition of the visceral fat, the data in

278

table 2 indicates higher deposits of retroperitoneal and epididymal fat in H and HP groups

279

when compared to group C.

280

Serum glucose disappearance rate (KITT) in obese mice decreased in relation to the control

281

mice, however, after treatment with pioglitazone, obese mice managed to reverse this,

282

reaching a percentage of decay similar to that of the control mice (KITT- C:

283

6.29±0.77%/min vs H: 2.69±0.30%/min vs HP: 4.85±0.60%/min; p<0.05). Therefore,

284

pioglitazone improved insulin sensitivity of obese animals (table 2 and Figure 1A). In

285

addition, animals of group H were hyperinsulinemic compared to group C, corroborating

286

with the insulin resistance observed. This effect was reversed in the HP group, which

287

presented plasma insulin levels similar to that of group C (table 2).

288 289

Pioglitazone increases oxidative capacity, fatty acid oxidation and glucose transport

290

in skeletal muscle of obese mice

291

We evaluated mRNA expression of genes involved in fatty acid oxidation and glucose

292

transport. Slc2a4 mRNA was induced by pioglitazone treatment of HFD mice (C:

293

1.00±0.24 vs H: 0.97±0.08 vs HP:1.76±0.16; p<0.05) (Figure 1B), corroborating with

294

pioglitazone improvements in observed insulin sensitivity (Table2). Pparg expression was

295

decreased in HFD-fed animals and pioglitazone treatment restored its expression (C:

296

1.00±0.03 vs H: 0.48±0.09 vs HP: 1.14±0.14; p<0.05) (Figure 1C). HFD promoted an

297

increased expression of Ppara which was exacerbated with pioglitazone treatment (C:

298

1.00±0.09 vs H: 3.33±0.44 vs HP: 9.23±1.25; p<0.05) (Figure 1D). There was no effect

299

of the diet in Cpt1b expression, however pioglitazone treatment increased its expression

300

when compared to H group (C: 1.00±0.15 vs H: 0.81±0.09 vs HP: 1.52±0.21; p<0.05)

13 301

(Figure 1E), correlating with increased fatty acid oxidation in skeletal muscle. In addition,

302

Ppargc1a and Ucp3 expression were reduced in soleus muscle of HFD-fed animals and

303

pioglitazone treatment restored its expression (Ppargc1a, C: 1.00±0.15 vs H: 0.60±0.06 vs

304

HP: 1.14±0.09 p<0.05; Ucp3, C: 1.00±0.13 vs H: 0.65±0.11 vs HP: 1.05±0.09; p<0.05)

305

(Figures 1F and 1G).

306

These results suggest that increased insulin-mediated glucose transport is a consequence of

307

increased fatty acid oxidation and mitochondrial activity. We analyzed the activity of

308

enzymes involved in glycolysis (pyruvate kinase, PK), Krebs cycle (citrate synthase, CS)

309

and β-oxidation (β-hydroxyacyl CoA dehydrogenase, BHAD) in gastrocnemius and soleus

310

muscles (Table 2).

311

We observed differences in PK and CS activity only in gastrocnemius muscle. PK activity

312

was increased by HFD and pioglitazone had no effect on restoring its activity, and CS

313

activity was increased by HFD and pioglitazone, suggesting an increased mitochondrial

314

activity (Table 2).

315

There was a significant increase in succinate dehydrogenase (SDH) activity after

316

pioglitazone treatment of HFD mice in soleus muscle (Figure 1H). Additionally, the

317

number of positive fibers for SDH (fiber stained darkly for SDH) was increased by

318

pioglitazone treatment, suggesting pioglitazone increases mitochondrial proliferation

319

(Figure 1H).

320

We also analyzed SDH activity in gastrocnemius muscle, but there was no difference in

321

the frequency of positive SDH stained fibers (supplementary Figure 1S).

322 323

Pioglitazone improvements in insulin sensitivity are mediated by PPARg

324

To better understand the mechanism by which pioglitazone increases insulin sensitivity,

325

we treated mice with pioglitazone and GW6471 or GW9662, a PPARa and PPARg

14 326

antagonist, respectively. Systemic and muscle insulin sensitivity was not impaired in

327

PPARa antagonist-treated mice, while it was not reversed by pioglitazone when PPARg

328

was blocked in obese mice (Figure 2A-C).

329

CS activity was measured in gastrocnemius muscle of mice treated with pioglitazone and

330

PPAR antagonists. CS activity increased in gastrocnemius muscle of pioglitazone and

331

PPARa antagonist treated mice, however the PPARg antagonist nullified the effect.

332

Altogether, our data indicates pioglitazone via PPARg induces mitochondrial activity and

333

restores insulin sensitivity in soleus muscle of obese mice.

334 335

Effect of pioglitazone on microRNAs in skeletal muscle of obese mice

336

We measured the expression of some microRNAs previously associated with obesity

337

(miR-23b, miR-221 and miR-222) in adipose tissue, soleus and gastrocnemius muscle.

338

The other reason to choose those microRNAs is the fact that predicted KEGG pathways

339

includes insulin and adipocytokine signaling.

340

After eight weeks of high-fat diet, miR-221 and miR-222 were up-regulated in adipose

341

tissue of HFD-fed mice and pioglitazone treatment only reversed miR-221 levels, similar

342

to control group (Figure 3). In contrast, while miR-221 expression in soleus muscle was

343

similar among the groups, miR-23b and miR-222 were up-regulated in H group compared

344

to C group, and treatment with pioglitazone was able to reverse this condition (Figures 3).

345

We also measure miR-23b and miR-222 expression in gastrocnemius muscle but it was

346

not significantly difference among the groups (supplementary Figure 1S). These data

347

support an effect of pioglitazone in soleus muscle, as previously described by Hallakou et

348

al. (1998).

15 349

To understand if miR-222-3p and miR-23b-3p regulation are dependent on PPARg, we

350

measured their expression in skeletal muscle of mice treated with PPARg antagonist.

351

PPARg blockage did not affect pioglitazone reduction of both microRNAs (Figure 3).

352

Next, we tested pioglitazone effects on microRNAs in muscle. To answer this question,

353

we treated C2C12 cells with palmitate and pioglitazone. While pioglitazone could reduce

354

miR-222 expression in C2C12 cells (Figure 4A), and miR-23b was increased by

355

palmitate, pioglitazone treatment could not restore miR-222 expression to control levels

356

(Figure 4B).

357

We next investigated in vitro, if inhibition of miR-222 or miR-23b could restore insulin

358

sensitivity of palmitate-induced insulin resistant C2C12 cells.

359

As shown in figure 4C, C2C12 cells treated with palmitate decreased pAKT Ser 473, a

360

marker of insulin sensitivity, and while inhibition of miR-23b reduced pAKT, inhibition of

361

miR-222 seems to increase insulin sensitivity in palmitate-treated C2C12 cells.

362

Cells overexpressing Pgc-1 alpha have increased fatty acid oxidative capacity and

363

increased mitochondrial biogenesis and respiration rates that results in increased energy

364

expenditure (Nikolic et al., 2012; St-Pierre et al., 2003).

365

We measured the expression of miR-23b and miR-222 in skeletal muscle cells

366

overexpressing Pgc-1 alpha to address if there is another condition in which the fatty acid

367

oxidative capacity of cells is increased and whether those microRNAs would be regulated.

368

A down-regulation of miR-222, but not miR-23b, was also observed in cells

369

overexpressing Pgc-1 alpha (Figure 4D).

370

Finally, we evaluated mitochondrial function in myotubes transfected with miR-23b or

371

miR-222 inhibitors and treated with palmitate using Seahorse analyzer (Figure 5). No

372

significant differences were observed in basal and ATP-linked mitochondrial oxygen

373

consumption rate (OCR), and proton leak in C2C12 cells treated with palmitate compared

16 374

to control-treated cells (Figure 5B-D). Mitochondrial oxidative capacity, as suggested by

375

mitochondrial reserve capacity (spare capacity), increases only in cells treated with

376

palmitate and miR-222 inhibitors, as compared to control cells (Figure 5E). Non-

377

mitochondrial respiration was calculated, and it was notably higher in palmitate-treated

378

cells compared to control cells. Inhibition of miR-23b could not prevent palmitate-induced

379

non-mitochondrial OCR, however inhibition of miR-222 protected cells from this increase

380

(Figure 5F).

381 382

Discussion

383

In this study, we demonstrated that: (1) pioglitazone increases mitochondrial activity in

384

skeletal muscle, as suggested by increased CS and SDH activity and mRNA expression of

385

genes related to fatty acid oxidation; (2) muscle miR-23b and miR-222 are significantly

386

up-regulated in diet-induced obese mice and pioglitazone corrects muscle miR-23b and

387

miR-222 levels in obese mice in a non-dependent PPARg mechanism; (3) inhibition of

388

miR-222 in part corrects insulin sensitivity and prevents increased non-mitochondrial

389

respiration in palmitate-treated muscle cells.

390

The 8-week protocol of HFD promoted obesity, which was associated with insulin

391

resistance. Pioglitazone treatment in the last 2 weeks restored hyperinsulinemia in HFD-

392

fed mice and also promoted an improvement on insulin sensitivity, which was observed in

393

similar protocols in other study (Kubota et al., 2006)

394

Pioglitazone treatment is known to induce weight gain mainly because of enhanced

395

adipocyte differentiation, promoting an increase in adipose tissue (de Souza et al., 2001;

396

Hermansen & Mortensen, 2007). Treatment with pioglitazone led to an increase in fat

397

deposition. This has already been observed in studies with pioglitazone and other drugs of

398

the thiazolidinedione class; it appears that the drug functions as a lipogenic and adipogenic

17 399

agent, leading the adipocytes to synthesize triacylglycerol and store lipids for possible

400

thermogenic activation (Burkey et al., 2000; Festuccia et al., 2009).

401

Although pioglitazone has a known effect on the decrease in triglyceride serum levels, but

402

without significant changes in low-density lipoprotein (LDL-C) and total cholesterol

403

(Betteridge, 2007), triglyceride serum levels were not decreased by pioglitazone, likely

404

because HFD did not promote any increase on this parameter compared to C group.

405

Although PPARg expression in skeletal muscle, comparing to adipose tissue, is considered

406

low (Loviscach et al., 2000), muscle PPARg knockout (MuPPARgKO) mice are insulin

407

resistant and have increased fat mass, suggesting a role of muscle PPARg for maintenance

408

of normal adiposity and insulin sensitivity (Norris et al., 2003).

409

In vitro, a study with C2C12 cells demonstrated that PPARg activation promotes an

410

increase in glucose uptake, which is impaired by pioglitazone treatment (Verma, Singh, &

411

Dey, 2004). Our results converge with these findings since pioglitazone increases insulin

412

sensitivity and blockage of PPARg with GW9662 inhibits pioglitazone insulin-sensitizer

413

effect, compared to HFD-fed mice.

414

GW9662 has been shown to be an adipogenic antagonist in vitro and in vivo. Treatment of

415

HFD mice with GW9662 appears to completely protect them from HFD-induced increases

416

in visceral adipose tissue mass, however it does not change HFD-induced glucose

417

intolerance (Nakano et al., 2006).

418

In this study, treatment with pioglitazone reverses the effects promoted by the high-fat

419

diet, including restoration of Ppargc1a expression to similar levels seen in control animals

420

and increased expression of CPT1b.

421

Regarding Ucp3 expression, studies have demonstrated that an overexpression of UCP3 in

422

muscle cells leads to both the decay of ROS levels and an increase in fatty acid oxidation

18 423

(MacLellan et al., 2005), and a single dose of pioglitazone is able to induce an increase in

424

Ucp3 expression in skeletal muscle of health rats (Brunmair et al., 2004).

425

Increased PPARa expression after pioglitazone treatment has been demonstrated in a study

426

in adipose tissue, in which this increase was associated with increased fatty acid oxidation

427

(Bogacka, Xie, Bray, & Smith, 2005). However, loss of PPARa has little to no impairment

428

on skeletal muscle lipid utilization gene regulation (Muoio et al., 2002). On the other

429

hand, Norris et al. have demonstrated MuPPARgKO mice have altered expression of

430

several lipid metabolism genes in the muscle (Norris et al., 2003). In our study,

431

mitochondrial activityinduced by pioglitazone, as suggested by CS activity, seems to be a

432

PPARg mediated effect. Pioglitazone has been already shown to improve oxidative

433

capacity of muscle in diabetic rats (Wessels et al., 2015) which is substantiated by our

434

data.

435

Because miRNAs are dysregulated under conditions of obesity, they have the potential to

436

serve as important mediators of metabolic crosstalk between different organs.

437

miR-222 expression has been found to be elevated in diabetic mice and in the plasma of

438

obese human patients (Chartoumpekis et al., 2012; Ortega et al., 2013; Ortega et al.,

439

2014). Interestingly, in 3T3-L1 differentiated adipocytes, miR-222 down-regulation

440

increases insulin-stimulated glucose uptake (Shi et al., 2014). Accordingly, we have

441

shown pioglitazone induces down-regulation of miR-222, which is associated with higher

442

insulin sensitivity. This seems to be a direct effect on skeletal muscle as suggested by in

443

vitro experiments in C2C12 cells.

444

Martins et al. (2018) have shown a decrease in O2 consumption was associated with an

445

increase in hydrogen peroxide production in the soleus muscle of animals fed a

446

HFD. Additionally, high levels of palmitate have been shown to cause mitochondrial

19 447

lipotoxicity, ROS production and cell death in skeletal muscle cells (Martins et al., 2012;

448

Tumova et al., 2016).

449

Importantly, miR-222 inhibition increases mitochondrial reserve capacity and reduces

450

palmitate-induced non-mitochondrial respiration, which is related to ROS production. This

451

protector effect of miR-222 may be related to a reduction in oxidative stress of C2C12

452

cells. Xue et al. (2015) have shown in human endothelial cells overexpression of miR-222

453

significantly induced intracellular ROS production via Pgc1a downregulation which

454

supports this hypothesis. Moreover, our results are in line with the possible protection of

455

miR-222 in insulin resistance promoted by palmitic acid, as increased ROS production is

456

associated with inhibition of insulin action (Meo et al., 2017).

457

Considering miR-23b, a previous study by our group has demonstrated that miR-23b

458

expression is increased in skeletal muscle of HFD-fed mice, and this was not reversed by

459

fenofibrate treatment, a PPARa agonist (Frias et al., 2018). The result obtained in the

460

present study confirms that this miR is increased in soleus muscle, but not in adipose

461

tissue, after feeding a HFD, and further that pioglitazone was able to reverse the increased

462

miR-23b expression. On the other hand, miR-23b down-regulation seems to not have a

463

direct effect on skeletal muscle, and may be secondary to pioglitazone effects on other

464

tissue. Corroborating with this hypothesis, in the liver of pioglitazone-treated mice an up

465

regulation of miR-23b has been found with was negatively correlated with steatosis score

466

(Mendonça et al., 2019).

467

Other microRNAs, as reviewed in Portius et al. 2017, have been demonstrated to be

468

regulated by PPARs nuclear receptors (Portius, Sobolewski, & Foti, 2017). Of note,

469

pioglitazone and rosiglitazone have been shown to modulate the expression of 27 different

470

miRNAs in human subcutaneous and visceral adipocytes (Dharap, Pokrzywa, Murali,

20 471

Kaimal, & Vemuganti, 2015; J. Yu et al., 2014). Our studies provide some evidence that

472

TZDs in skeletal muscle may also regulate microRNAs expression to promote their action.

473

In conclusion, pioglitazone improves mitochondrial activity in skeletal muscle, which in

474

turns improves insulin sensitivity. Moreover, miR-222 may be a novel PPARg-

475

independent mechanism through which pioglitazone improves insulin sensitivity in

476

skeletal muscle.

477 478

Funding

479

This publication was made possible due to a Young Investigator Grant from the Fundação

480

de Amparo à Pesquisa do Estado de São Paulo (FAPESP: 2011/05876-6) and a Regular

481

research grant (2015/24789-8) and additonal funding from Conselho Nacional de

482

Desenvolvimento Científico e Tecnológico (CNPq) to AR (471085/2013-8). MM was a

483

researcher fellow of FAPESP (2014/22046-5 and 2015/24650-0).

484 485

Acknowledgments

486

We thank professor William Festuccia, PhD for insightful discussions about the research,

487

Sidney Veríssimo Filho for technical support and Vitória de Mendonça for making the

488

graphical abstract figure. This study was financed in part by the Coordenação de

489

Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES) - finance Code 001. MM

490

and ES are recipients of FAPESP scholarships.

491 492

Conflict of interest statement

493

The authors declare that the research was conducted in the absence of any commercial or

494

financial relationships that could be considered as a potential conflict of interest.

495

21 496

Author contributions

497

Conceived and designed the research: AR, MM. Acquired, analyzed or interpreted data:

498

ACR, AOP, ARS, BAS, DSPSFG, ES, GMM, HNA, LRS, MM and TIL. Wrote the

499

manuscript: MM, and AR. Final revision: AR, AOP, ARS, BAS, DSPSFG, ES, GMM,

500

HNA, LRS, MM and TIL

501 502

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adiposity and insulin resistance but respond to thiazolidinediones, Journal of Clinical Investigation. 112, 608-618. Ortega FJ, Mercader JM, Catalan V, Moreno-Navarrete JM, Pueyo N, Sabater M, GomezAmbrosi J, Anglada R, Fernandez-Formoso JA, Ricart W, Fruhbeck G and Fernandez-Real JM (2013) Targeting the circulating microRNA signature of obesity. Clin Chem 59:781-92. doi: 10.1373/clinchem.2012.195776 Ortega FJ, Mercader JM, Moreno-Navarrete JM, Rovira O, Guerra E, Esteve E, Xifra G, Martinez C, Ricart W, Rieusset J, Rome S, Karczewska-Kupczewska M, Straczkowski M and Fernandez-Real JM (2014) Profiling of circulating microRNAs reveals common microRNAs linked to type 2 diabetes that change with insulin sensitization. Diabetes Care 37:1375-83. doi: 10.2337/dc13-1847 Phillips, S. A., Ciaraldi, T. P., Kong, A. P., Bandukwala, R., Aroda, V., Carter, L., . . . Henry, R. R. (2003). Modulation of circulating and adipose tissue adiponectin levels by antidiabetic therapy. Diabetes, 52(3), 667-674. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12606507 Portius, D., Sobolewski, C., & Foti, M. (2017). MicroRNAs-Dependent Regulation of PPARs in Metabolic Diseases and Cancers. PPAR Res, 2017, 7058424. doi:10.1155/2017/7058424 Rabol, R., Boushel, R., Almdal, T., Hansen, C. N., Ploug, T., Haugaard, S. B., . . . Dela, F. (2010). Opposite effects of pioglitazone and rosiglitazone on mitochondrial respiration in skeletal muscle of patients with type 2 diabetes. Diabetes Obes Metab, 12(9), 806-814. doi:10.1111/j.1463-1326.2010.01237. Shi Z, Zhao C, Guo X, Ding H, Cui Y, Shen R and Liu J (2014) Differential expression of microRNAs in omental adipose tissue from gestational diabetes mellitus subjects reveals miR-222 as a regulator of ERalpha expression in estrogen-induced insulin resistance. Endocrinology 155:1982-90. doi: 10.1210/en.2013-2046 St-Pierre, J., Lin, J., Krauss, S., Tarr, P. T., Yang, R., Newgard, C. B., & Spiegelman, B. M. (2003). Bioenergetic analysis of peroxisome proliferator-activated receptor gamma coactivators 1alpha and 1beta (PGC-1alpha and PGC-1beta) in muscle cells. J Biol Chem, 278(29), 26597-26603. doi:10.1074/jbc.M301850200 Tanaka, Y., Kita, S., Nishizawa, H., Fukuda, S., Fujishima, Y., Obata, Y., . . . Shimomura, I. (2019). Adiponectin promotes muscle regeneration through binding to Tcadherin. Sci Rep, 9(1), 16. doi:10.1038/s41598-018-37115-3 Tomas, E., Tsao, T. S., Saha, A. K., Murrey, H. E., Zhang Cc, C., Itani, S. I., . . . Ruderman, N. B. (2002). Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation. Proc Natl Acad Sci U S A, 99(25), 16309-16313. doi:10.1073/pnas.222657499 Tumova J, Andel M, Trnka J. (2016). Excess of free fatty acids as a cause of metabolic dysfunction in skeletal muscle. Physiol Res. 2016 65(2):193-207. Retrieved from http://www.biomed.cas.cz/physiolres/pdf/65/65_193.pdf Verma, N. K., Singh, J., & Dey, C. S. (2004). PPAR-gamma expression modulates insulin sensitivity in C2C12 skeletal muscle cells. Br J Pharmacol, 143(8), 1006-1013. doi:10.1038/sj.bjp.0706002 Wessels, B., Ciapaite, J., van den Broek, N. M., Houten, S. M., Nicolay, K., & Prompers, J. J. (2015). Pioglitazone treatment restores in vivo muscle oxidative capacity in a rat model of diabetes. Diabetes Obes Metab, 17(1), 52-60. doi:10.1111/dom.12388 Xu HE, Stanley TB, Montana VG, Lambert MH, Shearer BG, Cobb JE, McKee DD, Galardi CM, Plunket KD, Nolte RT, Parks DJ, Moore JT, Kliewer SA, Willson

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Figure Legends

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Figure 1: Pioglitazone restores insulin sensitivity by increasing fatty acid oxidation.

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C57BL/6J mice fed either a control (C) or high fat (H) diet and were treated with

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pioglitazone (HP). Insulin tolerance test curve (A); mRNA expression of Slc2a4 (B),

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Pparg (C), Ppara (D), Cpt1b (E), Ppargc1a (F), Ucp3 (G) was measured by real-time

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PCR and expression was normalized to 36b4 and Hprt1 genes. (H) Representative images

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of the soleus muscle sections after SDH staining and relative percentage of SDH positive

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fibers. Results are presented as mean±SEM (n=10/group). One-way ANOVA followed by

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Tukey´s post test was used for statistical analysis of the results. *# p<0.05: (*) vs C; (#) vs

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H.

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Figure 2: PPARg is required for pioglitazone insulin-sensitizer effect on skeletal

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muscle. C57BL/6J mice after feeding a HFD and treatment with pioglitazone in the

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absence or presence of GW6471 (PPARa antagonist) or GW9662 (PPARg antagonist);

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Insulin tolerance test (ITT) (A) and KITT obtained from ITT (B), Representative images of

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phospho-AKT (Ser473) and total AKT in soleus muscle stimulated or not stimulated with

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insulin measured by western blot. Expression was normalized to total protein content with

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Ponceau S. mean values obtained after analysis of pAKT in insulin-stimulated muscles,

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expressed as relative to control (C). Citrate synthase activity (D). Results are presented as

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mean ± SEM (n=10/group), analyzed by One-Way ANOVA followed by post test. *#&

798

p<0.05: (*) vs C; (#) vs H.

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Figure 3: Expression of miRNAs related to obesity in visceral adipose tissue and

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soleus skeletal muscle of C57BL/6J mice fed either control (C) or high-fat (H) diet

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and treated with pioglitazone (HP) in the absence or presence of GW6471 (PPARa

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antagonist) or GW9662 (PPARg antagonist). Results are presented as mean±SEM

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(n=12/group). One-way ANOVA followed by Tukey´s post-test was used for statistical

804

analysis of the results. *# p<0.05: (*) vs C; (#) vs H.

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Figure 4: Inhibition of miR-222 reverses insulin resistance induced by palmitate in

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C2C12 cells and is associated with an increased mitochondrial activity in cells

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overexpressing Pgc-1alpha. (A) miR-222 and (B) miR-23b were measured in control,

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palmitate and palmitate plus pioglitazone (pio)-treated C2C12 cells; (C) C2C12 cells were

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transfected with microRNA inhibitors and treated with palmitate (0.75mM) for 16h and

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stimulated with insulin (100nM) for 15 min for determination of phospho-AKT Ser

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473 expression. Gapdh was used as internal control. (*) vs control; (#) vs palmitate as

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indicated by One-Way ANOVA followed by Tukey´s post-test. Results are presented as

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mean±SEM from two experiments in triplicate. (D) microRNAs expression were

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quantified by real-time PCR in C2C12 cells overexpressing Pgc-1 alpha. *p<0.05 vs

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control pBabe as indicated by Student “t” test.

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Figure 5: Inhibition of miR-222 increases mitochondrial reserve capacity and non-

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mitochondrial respiration. (A) Oxygen consumption rates (OCR) corrected by non-

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mitochondrial OCR, (B-F) basal, ATP-linked, proton leak, spare capacity and non-

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mitochondrial OCR from C2C12 cells transfected with miR-23b or miR-222 inhibitor or

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scrambled control and treated with palmitate or vehicle for 16h. Values represent the mean

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± SEM. ∗P < 0.05 vs control as indicated by One-Way ANOVA followed by Tukey´s

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posttest.

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Table 1: Primers’ sequences used to quantify mRNA expression Gene

Forward Sequence

Reverse Sequence

AdipoQ

GAGAAAGGAGATGCAGGTCTTC

ACGCTGAGCGATACACATAAG

AdipoR1

GAAGATGGAGGAGTTCGTGTATAA

AGCAGGTAGTCGTTGTCTTTC

AdipoR2

AGGCTGGCTAATGCTTATGG

GATGTGGAAGAGCTGATGAGAG

Slc2a4

CATTCCCTGGTTCATTGTGG

GAAGACGTAAGGACCCATAGC

Ppara

TCGAATATGTGGGGACAAGG

TCTTGCAGCTCCGATCACAC

Pparg

CAAACCTGATGGCATTGTGAG

ATCTTAACTGCCGGATCCAC

Pgc1a

CACCAAACCCACAGAAAACAG

GGGTCAGAGGAAGAGATAAAGTTG

Cpt1b

CCTCCGAAAAGCACCAAAAC

GCTCCAGGGTTCAGAAAGTAC

Ucp3

GACTATGGATGCCTACAGAACC

ACTCCAGCAACTTCTCCTTG

Rplp0

TAAAGACTGGAGACAAGGTG

GTGTACTCAGTCTCCACAGA

Hprt1

CCTAAGATGAGCGCAAGTTGAA

CCACAGGACTAGAACACCTGCTAA

Table 2: Obesity features and metabolic parameters from C57BL/6J mice fed either control (C) or high fat (H) diet or treated with pioglitazone (HP). C

H

HP

Initial BW (g)

23.56±0.60

23.18±0.69

23.21±0.64

Final BW (g)

27.85±0.80

33.53±1.18*

33.32±1.25*

BW gain (g)

4.53±0.40

9.75±0.73*

10.31±0.65*

Retroperitoneal fat pad (g)

0.25±0.03

0.57±0.06*

0.52±0.06*

Epididymal fat pad (g)

0.75±0.08

1.49±0.16

1.53±0.17*

Mesenteric fat pad (g)

0.37±0.04

0.56±0.07*

0.68±0.10*

Brown adipose tissue (g)

0.12±0.01

0.11±0.01

0.22±0.02*#

Liver Weight (g)

1.14±0.05

1.10±0.04

1.10±0.05

Total Cholesterol (mg/dL)

140 ± 9

145 ± 13

122 ± 11

Triglycerides (mg/dL)

97 ± 5

71 ± 6

82 ± 6

HDL-C (mg/dL)

36 ± 3

37 ± 2

29 ± 1

LDL-C (mg/dL)

85 ± 9

86 ± 11

75 ± 7

VLDL-C (mg/dL)

19 ± 1

18 ± 1

16 ± 1

ALT (mg/dL)

8±1

7±1

7±1

Gastro PK (nmol.min-1 . mg of protein-1)

364.82±16.97 416.54±10.16* 401.99±13.89

Gastro BHAD (nmol.min-1 . mg of protein-1)

16.67±1.12

17.04±0.41

16.16±0.65

Gastro CS (nmol.min-1 . mg of protein-1)

67.20±4.59

122.19±5.31*

110.18±4.13*

Sol PK (nmol.min-1 . mg of protein-1)

282.74±4.89

251.62±7.47

255.6±4.03

Sol BHAD (nmol.min-1 . mg of protein-1) 16.26±0.86

14.93±0.58

14.83±0.81

Sol CS (nmol.min-1 . mg of protein-1)

374.47±19.65 385.05±23.99 415.77±20.76

KITT (%glucose/min)

6.29±0.77

2.69±0.30*

4.85±0.60#

Fasting Serum Insulin (ng/ml)

1.95±0.21

4.16±0.58*

2.75±0.24#

BW, body weight; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; VLDL, very-low-density lipoprotein cholesterol; ALT: alanine transaminase; KITT, plasma glucose disappearance rate of insulin tolerance test; Gastro: gastrocnemius muscle; CS, citrate synthase activity; BHAD, beta-hydroxy-acyl-coenzyme A dehydrogenase activity; PK, pyruvate kinase activity; Sol: soleus muscle. Results are presented as mean ± SEM (n=15/group), analyzed by One-Way ANOVA followed by Tukey’s post test. *# p<0.05: (*) vs C; (#) vs H.

Highlights

● Pioglitazone induces mitochondrial activity in skeletal muscle ● miR-222 is decreased by pioglitazone in DIO mice in a PPARgammaindependent manner ● miR-222 is possibly involved in pioglitazone reversal of insulin resistance ● miR-222 prevents increased non-mitochondrial OCR in palmitate-treated muscle cells.