Yang Wang

Gilles Fouret

Béatrice Bonafos

Agnieszka Blachnio-Zabielska

Thibault Leroy

David Crouzier

Bruno Barea

Sylvie Gaillet

Cédric Moro

Jérôme Lecomte

Beatrice Bonafos

Cedric Moro

Jerome Lecomte

Charles Coudray

Christine Feillet-Coudray

lipid accumulation, mitochondrial activity and oxidative stress and their relationship with impaired glucose homeostasis in high fat high fructose diet-fed rats

Keywords: Free radicals, High fat/high fructose diet, Impaired glucose homeostasis, Mitochondrial function, Muscle lipid composition, Muscle metabolism, Oxidative stress

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Metabolic syndrome components, including obesity, dyslipidemia and impaired glucose homeostasis, are increasing and become a major public health issue. Indeed, increased body weight becomes a major public health challenge in developed countries and increasingly in developing countries, where food supplies are abundant and physical activity is reduced [START_REF] Barness | Obesity: genetic, molecular, and environmental aspects[END_REF]. Obesity generally results from imbalanced energy intake and expenditure of the body from a metabolic point of view.

Skeletal muscle is an essential tissue for whole-body energy metabolism and is the predominant site of insulin-mediated glucose uptake in the post prandial state [START_REF] Defronzo | Skeletal muscle insulin resistance is the primary defect in type 2 diabetes[END_REF]. Skeletal muscle fatty acid metabolism is subject to extensive in vivo regulation, in particular by control of fatty acid entry into the cell, their transfer into the mitochondria and the capacity of the β-oxidation cycle [START_REF] Kraegen | Muscle insulin resistance: a case of fat overconsumption, not mitochondrial dysfunction[END_REF]. The balance between uptake and utilization of fatty acids will ultimately determine the magnitude of lipid accumulation in muscle cells. Consequently, high fat intake may lead to ectopic lipid deposition in muscle and metabolically active lipid intermediates that accumulate, such as diacylglycerols (DAG) and sphingolipids, in particular ceramides and sphingomyelin. These active/toxic lipids have been demonstrated to play essential roles in the establishment of impaired glucose homeostasis muscle and insulin resistance [START_REF] Holland | Sphingolipids, insulin resistance, and metabolic disease: new insights from in vivo manipulation of sphingolipid metabolism[END_REF][START_REF] Tonks | Skeletal muscle and plasma lipidomic signatures of insulin resistance and overweight/obesity in humans[END_REF]. Indeed, the high fat diet-stimulated sphingolipid formation contributes to dysregulated lipid accumulation, systemic insulin resistance, and cytokine expression and secretion from skeletal muscle and adipose tissues, exacerbating obesity-related physiopathological conditions.

In addition, many reports have indicated that some skeletal muscle phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and their fatty acyl composition, have also a role in insulin sensitivity regulation [START_REF] Borkman | The relation between insulin sensitivity and the fatty-acid composition of skeletal-muscle phospholipids[END_REF][START_REF] Riccardi | Dietary fat, insulin sensitivity and the metabolic syndrome[END_REF][START_REF] Clore | Skeletal muscle phosphatidylcholine fatty acids and insulin sensitivity in normal humans[END_REF][START_REF] Funai | Skeletal Muscle Phospholipid Metabolism Regulates Insulin Sensitivity and Contractile Function[END_REF][START_REF] Newsom | Skeletal muscle phosphatidylcholine and phosphatidylethanolamine are related to insulin sensitivity and respond to acute exercise in humans[END_REF].

Reactive oxygen species (ROS) are overproduced in obesity and the resulting oxidative damage could play a key role in the pathogenesis of impaired glucose homeostasis, diabetes and its complications [START_REF] Victor | Oxidative stress and mitochondrial dysfunction in type 2 diabetes[END_REF]. Mitochondria are one of the main cellular ROS generation sites and mitochondrial ROS production is closely associated with activity of the respiratory chain complexes [START_REF] Turrens | Mitochondrial formation of reactive oxygen species[END_REF][START_REF] Valko | Free radicals and antioxidants in normal physiological functions and human disease[END_REF]. The relationship between mitochondria and insulin action is highly complex [START_REF] Montgomery | Mitochondrial dysfunction and insulin resistance: an update[END_REF]. Several studies in various models of metabolic diseases or using genetically manipulated mice have reported a decrease in mitochondrial activity, while others have reported either no change or even an increase in mitochondrial oxidative metabolism in obese and insulin resistant humans and rodents [START_REF] Keung | Inhibition of carnitine palmitoyltransferase-1 activity alleviates insulin resistance in diet-induced obese mice[END_REF][START_REF] Ahn | Allyl isothiocyanate ameliorates insulin resistance through the regulation of mitochondrial function[END_REF][START_REF] Martins | Attenuation of obesity and insulin resistance by fish oil supplementation is associated with improved skeletal muscle mitochondrial function in mice fed a high-fat diet[END_REF].

Moreover, there is growing evidence that inflammation and oxidative stress are closely related, creating a vicious circle that may contribute to impaired glucose homeostasis installation [START_REF] Bai | Oxidative stress contributes to abnormal glucose metabolism and insulin sensitivity in two hyperlipidemia models[END_REF].

Undeniably, one question that comes up is when oxidative stress occurs in muscle tissue, and whether it contributes to, or is simply a consequence of, obesity and impaired glucose homeostasis installation and development in our obese animal model. Although some teams have addressed this issue, the obtained results are conflictual [START_REF] Das | Dietary fat proportionately enhances oxidative stress and glucose intolerance followed by impaired expression of the genes associated with mitochondrial biogenesis[END_REF][START_REF] Lionetti | Skeletal muscle subsarcolemmal mitochondrial dysfunction in high-fat fed rats exhibiting impaired glucose homeostasis[END_REF] and the question remains open.

This study aimed at determining the time-course of skeletal muscle lipid accumulation and composition as well as mitochondrial function and oxidative stress in parallel to the development of obesity and glucose intolerance in high fat/high fructose (HFHFr) diet-fed rats.

Materials and Methods

Animals and diets

Seventy five 6-weeks old male Wistar rats (Charles River, L'Arbresle, France) were housed (2 per cage) under conditions of constant temperature (20-22°C), humidity (45-50%) and a standard dark cycle (20.00-08.00 hours). The rats were randomized into two groups of animals and fed for 4 to 20 weeks one of the two following semi-purified diets: 1) control diet, or 2) high fat/high fructose diet (HFHFr). Seven rats from control group and 8 rats from HFHFr group were sacrificed after 4, 8, 12, 16 or 20 weeks of diet treatment.

The detailed composition of these two diets is given in the supplementary Table 1. Roughly, the control diet contains 4% lipids as soybean oil, and the HFHFr diet contains 35% lipids (1% soybean oil+8.5% lard+17% palm oil+8.5% cocoa butter and 0.2% cholesterol) and 25% fructose. As shown in the supplementary Table 2, the lipid fraction of the control diet was composed of 17.4% saturated fatty acids (SFA), 28.1% monounsaturated fatty acids (MUFA) and 54.5% polyunsaturated fatty acids (PUFA), and that of the HFHFr diet was composed of 44.2% SFA, 43.2% MUFA and 12.6% PUFA.

Rats were given free access to food and tap water. Rat body weight was determined weekly and food consumption was determined every two-three days. Our institution guidelines for the care and use of laboratory animals were observed and all experimental procedures were approved by the local ethical committee in Montpellier, France (Reference CEEA-LR-12002).

Oral glucose tolerance test

The oral glucose tolerance test (OGTT) was completed four to five days before rat sacrifice and performed as previously described [START_REF] Feillet-Coudray | The mitochondrial-targeted antioxidant MitoQ ameliorates metabolic syndrome features in 28 obesogenic diet-fed rats better than Apocynin or Allopurinol[END_REF]. Briefly, 6-hours fasting rats received by gavage 2.5 g glucose/kg body weight. Blood was sampled through the tail vein of conscious rats immediately prior to the gavage, and 20, 40, 60, 90, 120 and 180 min afterward and blood glucose was measured using glucose strips and a commercial glucometer (AccuChek Active, Roche Diagnostics, USA). The area under the curve (AUC) was calculated by the trapezium method [START_REF] Jenkins | Low glycemic index carbohydrate foods in the management of hyperlipidemia[END_REF]. The AUC values are expressed as g glucose/L/180 min.

Sampling and routine biochemical analyses

Four to five days after the OGTT, 9 hours-fasted rats (23h:00-08h:00) were anaesthetized with pentobarbital (40 mg/kg b.w.) (Ceva Santé Animale, Libourne, Fr) and blood was drawn from the abdominal artery with a heparinized syringe and distributed into dry and heparinized tubes. Blood tubes were centrifuged at 1000 g for 10 min at 4°C, plasma or serum were collected and stored at -80°C until analysis. Liver, heart, muscles, adipose tissue and kidney were removed, rinsed with 0.9% NaCl, weighed and kept at -80°C until analysis, except a gastrocnemius that has been used for the isolation of fresh mitochondria.

Muscle neutral lipids and diacylglycerols and ceramide species

A part of a gastrocnemius muscle was homogenized in NaCl (9 g/L) and Triton X-100 (0.1%) and free fatty acids (FFA), triglycerides (TG) and total cholesterol levels were quantified on the muscle homogenate by enzymatic methods with commercial kits (Wako-NEFA-C kit, Oxoid, Dardilly, France; Cholesterol RTU kit and triglycerides PAP kit, Biomérieux, Lyon, France).

The muscle diacylglycerols and ceramide contents were measured according to the methods described by Blachnio-Zabielska et al. [START_REF] Blachnio-Zabielska | A liquid chromatography/tandem mass spectrometry method for measuring the in vivo incorporation of plasma free fatty acids into intramyocellular ceramides in humans[END_REF][START_REF] Blachnio-Zabielska | Intramyocellular diacylglycerol concentrations and [U-(1)(3)C]palmitate isotopic enrichment measured by LC/MS/MS[END_REF]. Briefly, lipids were extracted from ∼20 mg of soleus muscle by the extraction mixture isopropanol/water/ethyl acetate (35/5/60; v/v/v). Quantitative measurement of ceramides and diacylglycerols was made using an Agilent 6460 triple quadrupole mass spectrometer. Both ceramides and diacylglycerols were analyzed using positive ion electrospray ionization source with multiple reaction monitoring. The chromatographic separation was performed using an Agilent 1290 Infinity Ultra High Performance Liquid Chromatography. A mixture of d17:1/8:0, d17:1/18:0, d17:1/18:1(9Z), d17:1/20:0, d17:1/24:0 and d17:1/24:1(15Z) was used as internal standards for ceramides and Deuterated DAG Mixture I and Mixture II were used as internal standards for diacylglycerols (Avanti Polar Lipids, Alabaster, AL, USA). Concentration of each compound was assessed against standard calibration curves.

Phospholipids analysis of gastrocnemius muscle

Lipids from gastrocnemius muscle were extracted by a Folch mixture of chloroform/methanol (2: PS, PE, PG, PA, CL respectively) were finally identified by comparing their retention factor (R f ) to authentic standards and quantified using calibration curves of the same standards [START_REF] Coudray | A mitochondrial-targeted ubiquinone modulates muscle lipid profile and improves mitochondrial respiration in obesogenic diet-fed rats[END_REF].

1 v/v)

Fatty acids composition of gastrocnemius muscle

An internal standard (12,5 µg C17:0/mL) was added to the Folch lipid extracts and fatty acid methyl esters were prepared by incubation with acidified methanol, according to the method of Lepage and Roy [START_REF] Lepage | Direct transesterification of all classes of lipids in a one-step reaction[END_REF]. Briefly, the methylation reagent was generated by mixing sulphuric acid with methanol and butylated hydroxytoluen as antioxidant (50 mg/L), and the sample was heated at 90°C for 45 min to get the fatty acid methyl esters. After addition of sodium bicarbonate, distilled water and hexane, the sample was vortexed, centrifuged, and the upper hexane layer was transferred to a glass vial, evaporated under a nitrogen stream at 37°C, and dissolved in 0.5 ml isooctane/ BHT for gas chromatography analysis. Individual fatty acid methyl esters were identified according to the retention times of standards by gas liquid chromatography with a Supelco Vax capillary column, an AS-3000 autosampler and flame-ionization detector (Thermo Fisher Scientific, Courtaboeuf, France). The fatty acid methyl esters were quantified using the chromatographic peak area according to the internal standard method.

Desaturase activity and unsaturation indices of gastrocnemius muscle

Because it is very tricky to directly measure the activity of the enzymes that catalyze the desaturation and elongation reactions in the fatty acid metabolic pathways, results in animals have supported the use of surrogate measures of desaturase activity such as use of desaturase indices [START_REF] Warensjo | Effects of saturated and unsaturated fatty acids on estimated desaturase activities during a controlled dietary intervention[END_REF].

These

Mitochondrial isolation and characteristics of gastrocnemius muscle

Mitochondrial isolation: muscle mitochondria were extemporaneously isolated by the differential centrifugation technique [START_REF] Aoun | Rat liver mitochondrial membrane characteristics and mitochondrial functions are more profoundly altered by dietary lipid quantity than by dietary lipid quality: effect of different nutritional lipid patterns[END_REF]. Briefly, a whole gastrocnemius muscle (2-3 g) was homogenized on ice (1 g wet tissue for 9 volumes of sucrose buffer: Sucrose 0.25 M, Tris 10 mM, EDTA 0.5 mM, pH 7.5) using a Polytron homogenizer. The homogenate was centrifuged at 900 g for 10 min at 4°C. The pellet was discarded and the supernatant was centrifuged at 10 000 g for 10 min at 4°C. The obtained mitochondrial pellet was suspended and washed with 2 ml of sucrose buffer and centrifuged at 10 000 g for 10 min at 4°C. The final mitochondrial pellet was suspended in 1 ml MIRO5 buffer (Sucrose 110 mM, Hepes 20 mM, KH2PO4 10 mM, Taurine 20 mM, K-Lactobionate 60 mM, MgCl 2 3 mM EGTA 0.5 mM, BSA 1 g/l, pH 7.1) and aliquoted for mitochondrial function measurements. Protein content was determined according to Bradford with bovine serum albumin as the standard [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF].

Membrane fluidity measurement: muscle mitochondria underwent three cycles of freezing/thawing to fragment and expose the mitochondrial membranes. The muscle mitochondrial membrane fluidity was assessed by electron spin resonance as previously described [START_REF] Aoun | Rat liver mitochondrial membrane characteristics and mitochondrial functions are more profoundly altered by dietary lipid quantity than by dietary lipid quality: effect of different nutritional lipid patterns[END_REF], using two spin labels: 5nitroxide stearate (5-NS) and 16-nitroxide stearate (16-NS). These both labels allow the measurement of the fluidity at both the surface of-and inside the lipid bilayer membrane of mitochondria, respectively.

Membrane potential measurement: mitochondrial membrane potential was monitored according to the method described by Baracca et al [START_REF] Baracca | Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F(0) during ATP synthesis[END_REF] using Rhodamine (RH-123) as a probe. Mitochondrial potential (∆ψ) changes have been evaluated by measuring RH-123 fluorescence quenching at the following steps: with RH-123 label alone (2 µM) in MIRO5 buffer plus muscle fresh mitochondria (0.25 mg proteins), plus glutamate/malate/succinate (2.5/5/5 mM), plus ADP (0.5 mM). RH-123 fluorescence was measured at 495 nm (excitation) and 525 nm (emission) under continuous stirring.

Mitochondrial oxygen consumption: mitochondrial respiration was determined by measuring mitochondrial oxygen consumption in two 2 ml air-tight thermo-stated chambers of high resolution Oxygraph (Oroboros Oxygraph 2k , Autriche) [START_REF] Aoun | Rat liver mitochondrial membrane characteristics and mitochondrial functions are more profoundly altered by dietary lipid quantity than by dietary lipid quality: effect of different nutritional lipid patterns[END_REF]. The chambers were equilibrated with the MIRO5 respiration buffer at 37°C and fresh mitochondria were then added (0.4 mg protein) followed by substrates malate/glutamate/succinate 2.5/5/5 mM (state 4) and ADP 0.5 mM (state 3). Respiratory coefficient ratio (RCR) was calculated as the ratio of state 3 to state 4 rates of respiration.

Mitochondrial ROS production: mitochondrial ROS production was measured on fresh mitochondria as previously described [START_REF] Barja | The quantitative measurement of H2O2 generation in isolated mitochondria[END_REF]. Briefly, reaction mixtures (2 ml), containing MIRO5 buffer, dihydrodichlorofluorescein (10 µM) and peroxidase (2 IU/ml), were incubated with 60 µg of mitochondrial proteins with or without substrates (malate/glutamate/succinate 2.5/5/5 mM, respectively) and with or without ADP (0.5 mM). Fluorescence was measured 60 minutes after reaction initiation at 490/525nm excitation and emission, respectively.

Mitochondrial enzymatic activities of gastrocnemius muscle

Citrate synthase (CS) activity was measured according to Srere [START_REF] Srere | Citrate synthase[END_REF]: the activity of the enzyme is measured by following the color of 5-thio-2-nitrobenzoic acid, which is generated from 5,5'-dithiobis-2-nitrobenzoic acid present in the reaction of citrate synthesis, and caused by the deacetylation of acetyl-CoA. The activity of the different mitochondrial respiratory complexes was determined as previously described [START_REF] Feillet-Coudray | Oxidative stress in rats fed a high-fat high-sucrose diet and preventive effect of polyphenols: Involvement of mitochondrial and NAD(P)H oxidase systems[END_REF]. Mitochondrial complex I activity was measured spectrophotometrically at 600 nm during 45 sec by following the reduction of 2,6-dichloroindophenol by electrons accepted from decylubiquinol, itself reduced after oxidation of NADH by complex I [START_REF] Janssen | Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts[END_REF]. Complex II (CII) activity was measured spectrophotometrically at 600 nm by following the reduction of 2,6dichloroindophenol by the succinate during 120 sec. Complex II+III (CII+CIII) activities were measured spectrophotometrically by following the reduction of cytochrome C at 550 nm during 90 sec [START_REF] Rustin | Biochemical and molecular investigations in respiratory chain deficiencies[END_REF]. Cytochrome c oxidase (CIV) activity was measured spectrophotometrically by following the oxidation of reduced cytochrome C at 550 nm during 30 sec [START_REF] Wharton | Cytochrome oxidase from beef heart mitochondria[END_REF].

In addition, mitochondrial β-hydroxyacyl-CoA dehydrogenase (β-HAD), a marker of last step of mitochondrial β-oxidation activity, was determined spectrophotometrically according to Clayton et al. [START_REF] Clayton | Hyperinsulinism in short-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency reveals the importance of beta-oxidation in insulin secretion[END_REF]. Briefly, muscle mitochondria suspension (25 µg protein) were mixed with NADH and acetoacetyl CoA in 1 ml of 50 mM Tris-HCl buffer, pH 7.0, and absorbance was followed at 340 nm for 60 seconds at 30°C.

Oxidative stress status of gastrocnemius muscle

A part of a gastrocnemius muscle was homogenized in phosphate buffer (50 mM, pH 7) 1 g for 9 ml buffer, using a Polytron homogenizer. The muscle thiobarbituric acid reactive substances (TBARS) and total glutathione (GSH) levels were measured in muscle homogenate according to Sunderman [START_REF] Sunderman | Increased lipid peroxidation in tissues of nickel chloride-treated rats[END_REF] and Griffith [START_REF] Griffith | Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine[END_REF], respectively. The remaining homogenate was centrifuged at 1000 g for 10 min at 4°C and the supernatant was used for the other analyses of oxidative stress. Protein oxidation was assessed in muscle supernatant by measurement of thiol groups as previously described [START_REF] Faure | Measurement of plasma sulfhydryl and carbonyl groups as a possible indicator of protein oxidation[END_REF]. Catalase activity was measured in muscle supernatant according to Beers and Sizer [START_REF] Beers | A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase[END_REF]. Glutathione peroxidase (GPx) was measured in muscle supernatant according to Flohe and Gunzler [START_REF] Flohe | Assays of glutathione peroxidase[END_REF]. Total superoxide dismutase (SOD) was measured in muscle supernatant according to Marklund [START_REF] Marklund | Spectrophotometric study of spontaneous disproportionation of superoxide anion radical and sensitive direct assay for superoxide dismutase[END_REF].

Protein isolation and western blotting analysis of gastrocnemius muscle

Frozen gastrocnemius muscle samples were homogenized using an Ultra Turax homogenizer in an ice cold extraction buffer containing 20 mM Tris-HCL, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X- RT-qPCR analysis are shown in the supplementary Table 4.

Statistical analysis

The sample size was calculated from the expected difference in the AUC of the OGTT between the control group and the HFHFr groups. We expected an AUC about 1.20 times greater in the HFHFr groups than in the control group. A standard deviation of 15% of the higher value could be allowed.

For a type 1 risk α of 0.05 and power (1-β) of 80%, the sample size required was eight per group.

Results were expressed as means±SD, n=7 animals per control group and 8 animals per HFHFr group per each treatment duration.

All the groups were tested for the effects of diet, treatment duration, and their interaction using a two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyze the effect of HFHFr diet compared to control diet within each diet treatment duration. The OGTT, mitochondrial characteristics parameters and the western blot analyses were carried out separately for each diet treatment duration. So statistical analysis for these parameters was made only between HFHFr and control diets, inside of each diet treatment duration, by the unpaired student's ttest. Correlations between parameters were performed with the Spearman method. The limit of statistical significance was set at p<0.05. HFHFr diet vs control diet: *p<0.05, **p<0.01, ***p<0.001.

Statistical analyses were performed using the StatView program (SAS Institute, Cary, NC, USA).

Results

Rat characteristics and blood biochemical parameters

Both dietary and energy intakes were increased throughout the study in both rat groups but the dietary intake was largely lower from the HFHFr diet compared to the control diet. Nevertheless, if the energy intake in both groups was overall similar, it was lower on 4 wks and higher on 12 and 16 wks from the HFHFr diet compared to the control diet (Table 1). In addition to a moderate but significant increase in body weight of HFHFr diet-fed rats (Table 1), the HFHFr rats exhibited higher liver and adipose tissue weights with lower gastrocnemius weight than control rats (Figure 1). These effects were further accentuated when expressed in relation to rat body weight (Figure 1). In addition, the two-way ANOVA analysis showed a slight but significant decrease in the weight of soleus, tibialis and extensor digitorum longus in the HFHFr rats compared to the control rats, although no significant difference was observed at any diet treatment duration between control and HFHFr rats. Finally, the heart and kidney weights remained unchanged (Table 1).

The time-course of OGTT for control and HFHFr rats is shown in Figure 2A and2Abis respectively. The AUC of the OGTT showed that impaired glucose homeostasis was increased in the HFHFr rats compared to the control rats, reaching a maximum at 12 wks of diet intake (Figure 2B).

Serum levels of both TG and total cholesterol were gradually and significantly increased in both diet groups whereas HFHFr diet had no significant effect on these parameters. Surprisingly, serum FFA levels were significantly decreased with the HFHFr diet compared to control diet (Figure 3).

Gastrocnemius muscle neutral lipids and soleus muscle DAG and ceramides contents

The gastrocnemius muscle levels of FFA and TG were increased with the HFHFr diet, whereas that of cholesterol remained unchanged (Figure 4A). Specifically, FFA and TG levels were significantly increased at 12 wks of HFHFr diet by comparison to control diet. Indeed, the muscle triglycerides content was highly increased (2.2 times increased compared to control rats at 12 wks of diet), whereas free fatty acids were only moderately increased (+24% increase compared to control rats at 12 wks of diet).

Total DAG content in the soleus muscle was increased since the 4th wk of diet with the HFHFr diet compared to controls. In particular, it was significantly increased at the16th wk of HFHFr diet by comparison to control diet (Figure 4B). All muscle DAG species C16-C16, C16-C18.1, C16-C18, C18.1-C18.1 and C18-C18.1 were significantly increased, except C16-C18.2 (decreased) and C18.1-18.2 (unchanged), with the HFHFr diet compared to the control diet (Table 2).

Total ceramide content in the soleus muscle was also increased with the HFHFr diet compared to controls. In particular, it was significantly increased at 4 and 12 wks of HFHFr diet by comparison to control diet (Figure 4B). This increase was due the increase in four important ceramide species, the sphingosine and sphinganine, as well as ceramides C18 and C20, despite the fact that the content of the five other ceramides, namely, C16, C18.1, C22, C24.1 and C24 remained unchanged (Table 3).

Because lipid droplets are the major cellular organelles for the storage of neutral lipids, we have investigated the gene expression level of key players involved in lipid droplets dynamics in the soleus, e.i., PLIN2, DGAT1, ATGL, CD36 and LPL. As shown in Figure 5, the two-way ANOVA test showed that only the gene expression of CD36 was significantly increased with the HFHFr diet compared to the control diet, particularly on the 16 th wk of diet. In addition, when only student's t-test was applied on the 16 th treatment duration, the gene expression of both PLIN2 and ATGL appeared also increased with the HFHFr diet compared to the control diet.

Phospholipids distribution and fatty acid composition in the gastrocnemius muscle

The muscle content of total phospholipids remained statistically similar with HFHFr and control diet. Individually, the percentage of different phospholipid classes remained also statistically similar with HFHFr and control diet (Supplementary Table 5). In particular, the percentage of cardiolipin, that is considered as a biomarker of mitochondrial content [START_REF] Larsen | Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects[END_REF], was not modified.

As expected, the percentage of muscle total SFA was increased under the HFHFr diet, which was due to the increase in both palmitic and stearic acid, except at 8 wks and 12 wks of diet where SFA percentage was unchanged between controls and HFHFr rats. (Figure 6, Table 4). In addition, the percentage of muscle total MUFAs was also increased with the HFHFr diet, in particular on 8 and 12 wks of diet intake (Figure 6). Indeed, the percentage of the minor MUFAs, namely, C16:1 n-7 and C18:1 n-7, was significantly decreased whereas that of the major MUFA, C18:1 n-9, has more than doubled (Table 4). Finally, the percentage of muscle total PUFA was markedly decreased under the HFHFr diet compared to the control diet (Figure 6). Specifically, the percentage of linoleic acid (18:2 n-6), a major PUFA, and docosahexaenoic acid (22:6n-3) was decreased whereas that of another major PUFA, arachidonic acid (20:4n-6), remained unchanged with the HFHFr diet compared to the control diet (Table 4).

Consequently, the unsaturation index of muscle fatty acids was decreased with the HFHFr diet compared to the control diet (Table 4). Indeed, this decrease in the unsaturation index was in agreement with the decrease in the percentage of PUFAs under the HFHFr diet compared to the control diet. In addition, the fatty acid desaturase indices of the three important desaturases (∆9 = [16:1n-7/16:0], ∆6 = [18:3n-6/18:2n-6] and ∆5 = [20:4n-6/20:3n-6]) were all decreased with the HFHFr diet compared to the control diet (Table 4). This is in agreement with the above reported decreases in the percentage of PUFAs and in the unsaturation index. Moreover, this was confirmed with gene expression of SCD1, the stearoyl-CoA desaturase (or ∆-9-desaturase) that catalyzes the ratelimiting step in the formation of oleate and palmitoleate from stearoyl-CoA and palmitoyl-CoA, respectively. In fact, SCD1 gene expression was significantly decreased with the HFHFr diet compared to the control diet (Table 4).

Lipid signalling pathways and mitochondrial activity in the gastrocnemius muscle

In order to explain the obtained results, we studied the muscle expression of several actors of lipid metabolism. To begin with the lipid uptake, the protein abundance of both FAT/CD36 and FATP1, two fatty acid transporters, remained unchanged (Tables 5), whereas the gene expression of FABP3, another fatty acid transporter, was increased with the HFHFr diet compared to the control diet (Table 6). Protein expression of the long chain acyl-CoA synthetase (ACSL1), that activates the conversion of 12 to 20 carbon fatty acids into acyl-CoA thioesters, remained unchanged, except on 12 wks of diet.

The muscle gene expression of PPARα, PPARγ and Srebp-1c, three key regulators of lipid metabolism, as well as the gene and protein expression of PGC-1a, a key regulator of energy metabolism, remained unchanged (Tables 5,6). In addition, protein abundance of mCPT-1, a mitochondrial fatty acids transporter, remained also unchanged. Regarding lipogenesis, the muscle protein abundance of total acetyl CoA carboxylase (ACC), a lipogenic enzyme providing the malonyl-CoA substrate for fatty acid synthesis and ser 79 p-ACC, its inactivated form, remained unchanged whereas protein abundance of muscle fatty acid synthase (FAS), a multifunctional enzyme that synthesizes palmitic acid, was significantly and markedly decreased all along the study with the HFHFr diet compared to the control diet (Table 5).

Mitochondrial characteristics and enzymatic activity in the gastrocnemius muscle

The main muscle gastrocnemius mitochondrial parameters characterising mitochondrial function, that is, membrane potential, membrane fluidity and respiration, remained unchanged in the HFHFr diet-fed rats compared to the control rats (Table 7). In addition, the enzymatic activities of citrate synthase, a renowned biomarker of mitochondrial content [START_REF] Larsen | Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects[END_REF], and mitochondrial respiratory chain complexes (complex I, complex II, complex II+II and complex IV) as well as β-hydroxybutyryl coenzyme A dehydrogenase (β-HAD), involved in fatty acid β-oxidation, remained unchanged with the HFHFr diet compared to the control diet (Table 8). Because cardiolipin play a key role in the mitochondrial bionenergetic, in particular in mitochondrial respiratory chain complexes assembly and activity, we have determined the gene expression and the protein abundance of the cardiolipin synthase (CLS) as well as the gene expression of the CDP-Diacylglycerol Synthase 1 (CDS1), a critical enzyme that catalyzes the formation of CDP-diacylglycerol from phosphatidic acid for de novo cardiolipin synthesis. The gene expression but not the protein abundance of CLS was slightly but significantly decreased with the HFHFr diet compared to the control diet, whereas the gene expression of CDS1 remained unchanged (Table 8).

Mitochondrial ROS production and oxidative stress status in the gastrocnemius muscle

Mitochondrial ROS production of gastrocnemius muscle challenged with substrates alone (malate+glutamate+succinate) or with substrates plus ADP, remained unchanged throughout the study between HFHFr and control rats (Table 9). In addition, several long-established oxidative and antioxidative markers were determined in this study. The gastrocnemius muscle levels of TBARS (marker of lipid peroxidation), sulfhydryl groups (marker of protein oxidation) and GSH (a major cellular antioxidant) as well as the enzymatic activities of the antioxidant enzymes SOD, catalase and GPx remained unchanged with the HFHFr diet compared to the control diet (Table 9).

Correlation of glucose intolerance with lipid composition, mitochondrial activity and oxidative stress parameters

The TG, cholesterol and FFA contents of gastrocnemius muscle were only slightly positively correlated with glucose intolerance. However, a moderate positive correlation was observed between soleus muscle DAGs content and glucose intolerance but not with soleus muscle ceramides content (Figure 7). Specifically, several major DAG species (C16/C18:1, C16/C18, C18:1/C18:1) were positively correlated with glucose intolerance. Regarding muscle FA composition, the muscle SFAs were not correlated whereas both MUFAs and PUFAs were respectively positively and negatively correlated with glucose intolerance (Figure 8). The determination of mitochondrial activity and ROS production showed that the enzymatic activities of the respiratory chain complexes I and IV were positively, whereas ROS production was negatively correlated with glucose intolerance (Figure 9). Finally, we observed no significant correlation between glucose intolerance and several measured oxidative stress parameters in the gastrocnemius muscle, namely, TBARS, sulfhydryl groups, GSH, SOD, catalase and GPx, monitored in this study (data not shown).

Discussion

It is well known that fatty acid composition of the diet can influence membrane fatty acid composition and then cellular activity and homeostasis [START_REF] Abbott | Membrane fatty acid composition of rat skeletal muscle is most responsive to the balance of dietary n-3 and n-6 PUFA[END_REF]. Indeed, many reports have shown that high intake of dietary saturated fat significantly impaired glucose homeostasis and thus worsen insulin-resistance [START_REF] Riccardi | Dietary fat, insulin sensitivity and the metabolic syndrome[END_REF]. However, changes over time in skeletal muscle lipid accumulation, mitochondrial function and oxidative stress, along with the onset of obesity and impaired glucose homeostasis, remain crucial and interesting questions in animal models of obesity. In this study, rats

were fed an experimental diet with a blend of fats with a high percentage of saturated fats, in particular palmitic acid. This diet was efficient in inducing moderate overweight, increasing liver and adipose tissue weights. These marked modifications of rat characteristics were clearly accompanied with muscle lipid accumulation and impaired glucose tolerance since the 8th week of diet.

Muscle lipid accumulated with HFHFr diet with a maximum at 12th week of diet

Muscle lipid accumulation and/or lipid metabolism alteration is known to play an important role in the development of glucose intolerance in HF feeding models [START_REF] Van Herpen | Lipid accumulation in non-adipose tissue and lipotoxicity[END_REF]. However, there is an ongoing debate on which of the lipid species plays the major role in inducing muscle insulin resistance and thus impaired glucose homeostasis. Although many studies have reported increased muscle lipid accumulation in mice/rats fed HF diets, it is the intracellular lipotoxic metabolites (fatty acyl CoA, DAG and ceramides) that are considered the true culprits responsible for the installation and development of muscle metabolic perturbations and thus impaired glucose homeostasis. Indeed, in addition to the increased muscle TG, cholesterol and FFA contents, our results indicated that muscle DAG and ceramides were markedly increased with the HFHFr diet compared to the control diet. This could result from fatty acid storage within lipid droplets of skeletal muscle reflected by a sustained higher gene expression of FAT/CD36 during the course of HFHFr diet. In addition, total DAG and several individual species (C16/C18 and C18:1/C18:1), as well as TG, cholesterol and FFA, were positively correlated with glucose intolerance in this study. In accordance, muscle DAG accumulation in both animals and humans has been linked to insulin resistance [START_REF] Finck | Does Diacylglycerol Accumulation in Fatty Liver Disease Cause Hepatic Insulin Resistance?[END_REF]. Although the cellular mechanisms underlying this relationship are still in debates, the connection between muscle DAG accumulation and insulin resistance could be attributed to activation of protein kinase C signaling [START_REF] Jornayvaz | Diacylglycerol activation of protein kinase Cepsilon and hepatic insulin resistance[END_REF].

In addition, ceramides are important second messengers that interact with several pathways involved in insulin resistance, oxidative stress, inflammation and apoptosis [START_REF] Pagadala | Role of ceramides in nonalcoholic fatty liver disease[END_REF]. Indeed, the ceramides have been shown to inhibit several distinct intermediates in the insulin signaling pathway, by regulating IRS-1, PI3-kinase, and Akt/PKB [START_REF] Summers | Ceramides in insulin resistance and lipotoxicity[END_REF]. It was shown that the muscle ceramide (C18:1/C18:0) was the only higher lipid in insulin resistance independent of overweight/obesity [START_REF] Blachnio-Zabielska | The Crucial Role of C18-Cer in Fat-Induced Skeletal Muscle Insulin Resistance[END_REF]. In addition, muscle insulin resistance was predominantly associated with higher C18:0 ceramide [START_REF] Tonks | Skeletal muscle and plasma lipidomic signatures of insulin resistance and overweight/obesity in humans[END_REF]. However, although the accumulation of muscle stearoyl-ceramide species (C18:0) is considered having the greatest impact on skeletal muscle insulin sensitivity [START_REF] Blachnio-Zabielska | The Crucial Role of C18-Cer in Fat-Induced Skeletal Muscle Insulin Resistance[END_REF], no ceramide species was correlated with impaired glucose homeostasis in the present study.

Interestingly, maximal lipid accumulation under the HFHFr diet was observed at 12 th /16 th wk of diet. This is particularly notable for TG and DAG, but also for cholesterol and FFA. This was associated with higher gene expression of muscle FAT/CD36, a major player of cellular lipid storage and trafficking. In accordance, other parameters were modified maximally at this time of the diet, in particular OGTT, plasma glucose and HOMA-IR index, reflecting maximal impaired glucose tolerance. A previous study from our laboratory also demonstrated that hepatic steatosis and many biochemical modifications plateaued at 8-12 weeks of HFHFR diet in rats with slight amelioration afterwards [START_REF] Fouret | 20-Week follow-up of hepatic steatosis installation and liver mitochondrial structure and activity and their interrelation in rats fed a high-fat-high-fructose diet[END_REF]. It is likely that adaptive mechanisms are initiated to attenuate the effect of the HFHFr diet after some weeks of diet. However, none of the proteins from the lipid metabolism signaling pathways we have explored has allowed us to explain such observation.

Muscle fatty acid composition was altered as soon as 4 weeks of HFHFr diet

The fatty acid composition of the gastrocnemius muscle was modified as early as 4 weeks of diet.

The percentage of both major SFAs, C16 and C18, was increased with HFHFr diet compared to the control diet. However, there was no significant correlation between total SFA or individual SFAs with glucose intolerance. The intake of the HFHFr diet led also to an overall increase in muscle MUFA, in particular the major MUFA, C18:1 n-9. This results from the dietary C18:1 n-9 and not from in vivo conversion of saturated fatty acids into monounsaturated fatty acids, which serve as the preferred substrate for synthesis of stored lipids [START_REF] Dobrzyn | The role of stearoyl-CoA desaturase in the control of metabolism[END_REF][START_REF] Dobrzyn | Stearoyl-CoA desaturase and insulin signaling--what is the molecular switch?[END_REF], as SCD1 gene and activity were decreased with the HFHFr diet. Such a result may contradict the idea that MUFAs are rather beneficial in improving insulin sensitivity compared to a high-saturated-fat diet [START_REF] Riccardi | Dietary treatment of the metabolic syndrome--the optimal diet[END_REF]. Another consequence of this HFHFr diet was a significant decrease in the percentage of muscle PUFAs, in particular in the metabolically important linoleic acid whereas the arachidonic acid percent remained unchanged. Interestingly, the muscle total PUFA as well as many of individuals PUFAs, among which n-6 and n-3 PUFA, were negatively correlated with glucose intolerance. This observation reinforces the concept that PUFAs are beneficial for insulin sensitivity [START_REF] Riserus | Fatty acids and insulin sensitivity[END_REF]. Indeed, diets rich in SFAs induce impaired glucose homeostasis with concomitant increases in muscle DAG whereas diets rich in PUFAs appear to prevent glucose intolerance by directing fat into TG formation rather than in other toxic lipid metabolites [START_REF] Lee | Saturated, but not n-6 polyunsaturated, fatty acids induce insulin resistance: role of intramuscular accumulation of lipid metabolites[END_REF].

Dysfunction of the muscle mitochondria is not mainly involved in glucose intolerance

Skeletal muscle mitochondria may play a key role in high fat or HF/HFr diet induced insulin resistance [START_REF] Lark | High-fat load: mechanism(s) of insulin resistance in skeletal muscle[END_REF]. Two mechanisms by which mitochondrial function could be involved in skeletal muscle IR are debated [START_REF] Putti | Skeletal Muscle Mitochondrial Bioenergetics and Morphology in High Fat Diet Induced Obesity and Insulin Resistance: Focus on Dietary Fat Source[END_REF]. The first suggests that mitochondrial dysfunction causes intramyocellular lipid accumulation leading to IR, while the second involves excess fuel within mitochondria in the absence of increased energy demand, stimulating mitochondrial oxidant production and emission, and ultimately leading to the development of IR. The first mechanism may be more privileged but not in a significant way as both the content and characteristics of muscle mitochondria and mitochondrial activity remained unchanged in this study. However, the study of mitochondrial activity showed that the enzymatic activities of respiratory chain complexes I and IV were positively correlated with impaired glucose homeostasis, probably indicating that mitochondrial activity was impacted by the HFHFr diet although this was not appearing with the statistical analysis between the control group and the HFHFr group. Moreover, our results do not exclude possible important modifications of mitochondrial content and function in other tissues, in particular pancreas. In agreement with our results, Holloszy [START_REF] Holloszy | Deficiency" of mitochondria in muscle does not cause insulin resistance[END_REF] has reported that the observed reduction in muscle content of mitochondria in patients with types 2 diabetes is not responsible for insulin resistance.

The other mechanism involving production and release of mitochondrial oxidants that may induce insulin resistance is not supported by our present results. Indeed, the muscle mitochondrial ROS production was not modified by the HFHFr diet. In addition, the muscle oxidative and anti-oxidative stress parameters remained unchanged under the HFHFr diet compared to the control diet and this throughout the study. ROS production from muscle mitochondria were even negatively correlated with impaired glucose homeostasis. These results are consistent with many of our earlier findings of marked oxidative stress in the blood and/or liver, but not in skeletal muscle with HFHFr diet [START_REF] Fouret | 20-Week follow-up of hepatic steatosis installation and liver mitochondrial structure and activity and their interrelation in rats fed a high-fat-high-fructose diet[END_REF][START_REF] Feillet-Coudray | Effects of long-term administration of saturated and n-3 fatty acid-rich diets on lipid utilisation and oxidative stress in rat liver and muscle tissues[END_REF].

Moreover, many studies have reported oxidative stress onset in liver, intestine, adipose tissue or pancreas but rarely in skeletal muscle and/or heart in rats fed high-caloric diets [START_REF] Johnson | Cafeteria diet-induced obesity causes oxidative damage in white adipose[END_REF][START_REF] Pirozzi | Hydroxytyrosol prevents metabolic impairment reducing hepatic inflammation and restoring duodenal integrity in a rat model of NAFLD[END_REF]. In line with this and in a very nice proteomic study, Mendez et al [START_REF] Mendez | Protein carbonylation associated to high-fat, high-sucrose diet and its metabolic effects[END_REF] have reported that long-term HFHS diet consumption increased protein oxidation in plasma and liver; meanwhile protein carbonyls from skeletal muscle did not change. Unlike the liver, skeletal muscle is a second target of nutrients, and is therefore less exposed than the liver to direct dietary changes, including high calorie diets.

Conclusion

The present results indicate that muscle lipid alterations, as well as impaired glucose homeostasis occur as early as the 4-8th week of HFHFr diet, increase to reach a plateau around the 12 th weeks, and then attenuate towards the end of study (20 th weeks). In addition, at these diet treatment durations, muscle mitochondrial activity and oxidative stress remained unchanged and do not seem to play a major role in the development of glucose intolerance, while accumulation of lipids and intracellular lipotoxic metabolites may rather be involved with probable species specificity. Results were expressed as means ± SD, n=6-8 animals per group per diet treatment duration, rats liver (A), adipose tissue (B) and gastrocnemius (C) weights and their ratio to rat body weight. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each treatment duration. The limit of statistical significance was set at p<0.05. HFHFr diet vs control diet: *p<0.05, **p<0.01, ***p<0.001.

Figure 2: Time-course of oral glucose tolerance test in rats fed control or HFHFr diets for 4-20 weeks

A period of 6-h fasting rats received by gavage 2.5 g glucose/kg body weight. Blood was sampled through the tail vein of conscious rats immediately before the gavage, and 20, 40, 60, 90, 120 and 180 min afterwards and blood glucose was measured using glucose strips and a commercial glucometer.

Changes in blood glucose after oral glucose load are shown for control (A) and HFHFr (Abis) rat groups. Total cumulative glucose levels for each treatment group are reported as AUC and were calculated by the trapezium method and expressed as g glucose/l per 180 min (B). Results were expressed as means ± SD, n=6-8 animals per group per diet treatment duration. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each treatment duration. The limit of statistical significance was set at p<0.05. HFHFr diet vs control diet: *p<0.05, **p<0.01, ***p<0.001. AUC: area under the curve; OGTT: oral glucose tolerance test. Correlations were performed with the Spearman method. Glucose tolerance test was negatively correlated with muscle TG levels but positively correlated with muscle DAG levels. AUC: area under the curve; DAG: diacyglycerols; TG: triglycerides.

Figure 8: Spearman correlations between glucose tolerance test and gastrocnemius muscle fatty acid composition

Results were expressed as means ± SD, n=6-8 animals per group per diet treatment duration.

Correlations were performed with the Spearman method. AUC: area under the curve; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

Figure 9: Spearman correlations between glucose tolerance test and mitochondrial activity markers in the gastrocnemius muscle

Results were expressed as means ± SD, n=6-8 animals per group per diet treatment duration.

Correlations were performed with the Spearman method. ADP: adenosine diphosphate; AUC: area under the curve; CI: complex I; COX: complex IV/cytochrome oxidase; MGS: malate/glutamate/succinate; ROS: reactive oxygen species. 1 Table 1: Food consumption and rat body and organ weights.

Results were expressed as means±SD, n=6-8 animals per group per diet treatment duration. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each diet treatment duration. The limit of statistical significance was set at p<0.05. HFHFr diet vs control diet: *p<0.05, **p<0.01, ***p<0.001. NS: not significant. EDL: extensor digitorum longus; ww: wet weight. Results were expressed as means±SD, n=6-8 animals per group per diet treatment duration. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each diet treatment duration. The limit of statistical significance was set at p<0.05. HFHFr diet vs control diet: *p<0.05, **p<0.01. NS: not significant. ww: wet weight. The WB results were obtained as blot intensity of the studied protein divided by blot intensity of β-actin and normalised by the blot intensity of one control reference in the same gel: (protein blot intensity/actin blot intensity)/(protein blot intensity/actin blot intensity) control reference . Then all the results were normalized by the mean value obtained for the control group inside of each diet treatment duration. Results were expressed as means±SD, n=6-8 animals per group per diet treatment duration. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each diet treatment duration. The limit of statistical significance was set at p<0.05. NS: not significant. ADP: adenosine diphosphate; AU: arbitray units; MGS: malate/glutamate/succinate; 5-NS: 5-nitroxide stearate; 16-NS, 16nitroxide stearate; RCR: respiratory coefficient ratio. 8: Gastrocnemius mitochondrial respiratory chain complexes activities and cardiolipin synthesis Results were expressed as means±SD, n=6-8 animals per group per diet treatment duration. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each treatment duration. Because western blotting assay of CLS was performed separately on each diet treatment duration, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each diet treatment duration. The limit of statistical significance was set at p<0.05. NS: not significant. CDS1: CDP-diacylglycerol synthase 1; CLS: cardiolipin synthase; β-HAD: β-hydroxyacyl coenzyme A dehydrogenase; qPCR: quantitativepolymerase chain reaction; WB: western blotting.

  indices are based on the ratio product to precursor of individual fatty acids. The desaturase indices were calculated as follows: ∆9 desaturase = [16:1 n-7/16:0], ∆6 desaturase = [18:3 n-6/18:2 n-6] and ∆5 desaturase = [20:4 (n-6)/20:3 (n-6)]. In addition, we have determined the gene expression of the stearoyl-CoA desaturase 1 (SCD1= ∆9 desaturase) in the muscle. The unsaturation index (UI) was calculated from the relative percentage of each type of mono-and polyunsaturated fatty acid multiplied for the number of double bonds present in the molecule [29].

  triglyceride lipase; AUC: area under the curve; CDP: Cytidine diphosphate; CL: cardiolipin; CLS: cardiolipin synthase; DGAT1: diacylglycerol O-acyltransferase 1; FAS: fatty acid synthase; FAT/CD36: cluster of differentiation 36 (fatty acid translocase); FATP1: fatty-acid-transporter protein 1; GPx: Glutathione peroxidase; GSH: Glutathione; β-HAD: β-hydroxyacyl-CoA dehydrogenase; HFHFr: high fat high fructose; HPTLC: High performance thin layer chromatography; LPC: lysophosphatidylcholine; LPL: lipoprotein lipase; m-CPT1: muscle-Carnitine palmitoyltransferase I; MUFA: monounsaturated fatty acids; NAFLD: non-alcoholic fatty liver disease; 5-NS: 5-nitroxide stearate; 16-NS, 16-nitroxide stearate; OGTT: oral glucose tolerance test; PA: phosphatidic acid; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PG: phosphatidylglycerol; PGC-1α: peroxisome proliferator-activated receptor-gamma coactivator-1α; PI: phosphatidylinositol; PLIN2: perilipin 2; PS: phosphatidyl-serine; PUFA: polyunsaturated fatty acids; Rf: retention factor; qPCR: quantitativepolymerase chain reaction; RNS: reactive nitrogen species; ROS: reactive oxygen species; SCD1: stearoyl-CoA desaturase 1; SFA: saturated fatty acids; SM: sphingomyelin; SOD: superoxide dismutase; TBARS: Thiobarbituric acid reactive substances; TLC: thin layer chromatography; WB: western blotting.

Figure 1 :

 1 Figure 1: Weight of liver, adipose tissue and gastrocnemius and their ratios to rat body weight in rats fed control or HFHFr diets for 4 to 20 weeks

Figure 3 :

 3 Figure 3: Blood lipid levels in rats fed control or HFHFr diets for 4 to 20 weeksResults were expressed as means ± SD, n=6-8 animals per group per diet treatment duration. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each treatment duration. The limit of statistical significance was set at p<0.05. HFHFr diet vs control diet: *p<0.05, **p<0.01, ***p<0.001.

Figure 4 :

 4 Figure 4: Muscle neutral lipid accumulation in rats fed control or HFHFr diets for 4 to 20 weeksResults were expressed as means ± SD, n=6-8 animals per group per diet treatment duration. Muscle gastrocnemius TG, cholesterol and FFA (A), and muscle soleus DAG and Ceramides putative mediators of lipid-induced hepatic insulin resistance (B). All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each

Figure 5 :

 5 Figure 5: Gene expression of the major players in the formation of lipid droplets in the soleus muscle in rats fed control or HFHFr diets for 4 to 20 weeks Results were expressed as means ± SD, n=6-8 animals per group per diet treatment duration. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each treatment duration. The limit of statistical significance was set at p<0.05. HFHFr diet vs control diet: *p<0.05, **p<0.01, ***p<0.001. ATGL: adipose triglyceride lipase; CD36: cluster of differentiation 36 (fatty acid translocase); DGAT1: diacylglycerol Oacyltransferase 1; LPL: lipoprotein lipase; PLIN2: perilipin 2.

Figure 6 :

 6 Figure 6: Fatty acid classes in the gastrocnemius muscle from rats fed control or HFHFr diets for 4 to 20 weeksResults were expressed as means ± SD, n=6-8 animals per group per diet treatment duration. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each treatment duration. The limit of statistical significance was set at p<0.05. HFHFr diet vs control diet: *p<0.05, **p<0.01, ***p<0.001. MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; SFA: saturated fatty acids.

Figure 7 :

 7 Figure 7: Spearman correlations between glucose tolerance test and accumulation of key lipids in gastrocnemius and soleus musclesResults were expressed as means ± SD, n=6-8 animals per group per diet treatment duration.

Figure 1 :Figure 2 :

 12 Figure 1: Weight of liver, adipose tissue and gastrocnemius and their ratios to rat body weight in rats fed control or HFHFr diets for 4 to 20 weeks

Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :

 345678 Figure 3: Blood lipid levels in rats fed control or HFHFr diets for 4 to 20 weeks

Figure 9 :

 9 Figure 9: Spearman correlations between glucose tolerance test and mitochondrial activity markers in the gastrocnemius muscle

3 : 4 :

 34 Content of ceramides species in the soleus muscle (µg/g ww)Results were expressed as means±SD, n=6-8 animals per group per diet treatment duration. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each diet treatment duration. The limit of statistical significance was set at p<0.05. HFHFr diet vs control diet: *p<0.05, **p<0.01, ***p<0.001. NS: not significant. ww: wet weight. Fatty acid composition (%) of muscle lipids in the gastrocnemius muscle Results were expressed as means±SD, n=6-8 animals per group per diet treatment duration. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each diet treatment duration. The limit of statistical significance was set at p<0.05. HFHFr diet vs control diet: *p<0.05, **p<0.01, ***p<0.001. NS: not significant. MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; qPCR: quantitative-polymerase chain reaction; SCD1: stearoyl-CoA desaturase 1; SFA: saturated fatty acids. Unsaturation index = (% monoenoic x1) + (% dienoic x2) + (% trienoic x3) + (% tetraenoic x4) + (% pentaenoic x5) + (% hexaenoic x6).

Table 5 :

 5 Protein abundance of major actors of lipid metabolism in the gastrocnemius muscleResults were expressed as means±SD, n=6-8 animals per group per diet treatment duration. Because western blotting assay was performed separately on each treatment duration, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each diet treatment duration. The limit of statistical significance was set at p<0.05. *p<0.05. ACC: acetyl-CoA carboxylase; ACSL1: acyl-CoA synthetase long chain family member 1; CD36/FAT: cluster of differentiation 36 (fatty acid translocase); FAS: fatty acid synthase; FATP1: fatty-acidtransporter protein; mCPT1: muscle carnitine palmitoyltransferase I; PGC-1α: peroxisome proliferator-activated receptor g coactivator-1α.

9 Table 9 :

 99 ROS production and oxidative stress parameters in the gastrocnemius muscleResults were expressed as means ± SD, n=6-8 animals per group per diet treatment duration. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA test. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each diet treatment duration. The limit of statistical significance was set at p<0.05. NS: not significant. ADP: adenosine diphosphate; GSH: glutathione; GPx: glutathione peroxidase; MGS: malate/glutamate/succinate; SOD: superoxide dismutase; TBARS: thiobarbituric acid reactive substances; Thiols: sulfhydryl groups; ww:

Table 2 :

 2 Content of diacylglycerols species in the soleus muscle (µg/g ww)

Table 6 :

 6 Gene expression of major actors of lipid metabolism in the gastrocnemius muscleResults were expressed as means±SD, n=6-8 animals per group per diet treatment duration. All the groups were tested for the effects of diet, treatment duration, and their interaction by two-way ANOVA. When the effect of diet was significant, the unpaired student's t-test was used to analyse the effect of HFHFr diet within each diet treatment duration. The limit of statistical significance was set at *p<0.05. NS: not significant. FABP3: fatty-

	Parameters	Diet	4 wks	8 wks	12 wks	16 wks	20 wks
	CD36/β-actin	Control	1.00±0.17	1.00±0.15	1.00±0.36	1.00±0.36	1.00±0.12
		HFHFr	1.09±0.34	1.20±0.47	0.97±0.39	0.85±0.25	0.85±0.26
	FATP1/β-actin	Control	1.00±0.20	1.00±0.8	1.00±0.16	1.00±0.18	1.00±0.70
		HFHFr	0.97±0.38	1.04±0.20	1.24±0.33	1.20±0.39	0.54±0.41
	ACSL1/β-actin	Control	1.00±0.28	1.00±0.21	1.00±0.16	1.00±0.20	1.00±0.12
		HFHFr	0.89±0.16	0.82±0.30	1.39±0.38*	1.05±0.39	1.10±0.31
	PGC1a/β-actin	Control	1.00±0.12	1.00±0.11	1.00±0.30	1.00±0.32	1.00±0.76
		HFHFr	0.99±0.18	0.79±0.35	1.34±0.47	0.77±0.32	0.81±0.48
	mCPT1/β-actin	Control	1.00±0.27	1.00±0.37	1.00±0.28	1.00±0.09	1.00±0.22
		HFHFr	0.98±0.24	1.04±0.38	0.90±0.38	1.10±0.44	0.91±0.16
	ACC/β-actin	Control	1.00±0.07	1.00±0.07	1.00±0.24	1.00±0.36	1.00±0.33
		HFHFr	0.88±0.21	0.84±0.27	1.27±0.30	0.93±0.35	1.07±0.18
	p-ACC/β-actin	Control	1.00±0.36	1.00±0.37	1.00±0.35	1.00±55	1.00±0.39
		HFHFr	0.99±0.38	0.63±0.32*	1.20±0.25	0.89±0.41	0.96±0.55
	FAS/β-actin	Control	100±0.73	1.00±0.55	1.00±1.27	1.00±1.29	1.00±0.35
		HFHFr	0.68±0.35	0.36±0.35*	0.18±0.16	0.50±0.27	0.58±0.46*

acid-binding protein 3; PGC-1α: peroxisome proliferator-activated receptor g coactivator-1α; PPARα: peroxisome proliferator activated receptor alpha; PPARγ: peroxisome proliferator activated receptor gamma; Srebp-1c: Sterol regulatory element-binding transcription factor 1c. E: efficiency of the gene and C(t): cycle threshold obtained by relative qPCR. Then all the results were normalized by the mean value obtained for the control group at 4 weeks. In such a case, all the other groups were relative to the control group at 4 weeks of diet.

Table 7 :

 7 Characterization of mitochondrial functionality in the gastrocnemius muscle

Acknowledgments

The authors acknowledge the financial support of the National Institute for Agronomic Research and in particular the Human nutrition Department (Alim-H department). The authors wish also to thank the animal staff from Metamus DMEM facility which belongs to Montpellier animal facilities network (RAM, Biocampus).

Funding

Yang Wang thanks for the support from PhD program of the China Scholarship Council.

Author contributions

Author contributions were as follows: study design (CC, CF-C), data collection (YW, GF, BB, AB-Z, BB, JL, TL, CM), statistical analysis (CC, CF-C), data interpretation (CC, CF-C, SG), manuscript preparation (CC, CF-C, CM), literature search (CC & CF-C) and funds collection (CF-C).

Conflict of interest

The authors have no conflicts of interest to declare.