Demangel, R Anthony Damiot

Rémi Demangel

John Noone

Isabelle Chery

Alexandre Zahariev

Sylvain Normand

Thomas Brioche

Francois Crampes

Isabelle De

Sylvie Normand

François Crampes

Isabelle De Glisezinski

Etienne Lefai

Marie Pierre Bareille

Angèle Chopard

Jocelyne Drai

Delphine Collin-Chavagnac

Martina Heer

Guillemette Gauquelin-Koch

Michel Prost

Patrick Simon

Guillaume Py

Stéphane Blanc
email: stephane.blanc@iphc.cnrs.fr

Chantal Simon

Audrey Bergouignan

Donal J O'gorman

De Glisezinski

A nutrient cocktail prevents lipid metabolism alterations induced by 20 days of daily steps reduction and fructose overfeeding: Result from a randomized study

Keywords: nutrition, physical inactivity, sedentary behaviors, countermeasure, polyphenols, omega-3 fatty acids, selenium, vitamins, OGTT Akt: Protein kinase B AUC: Area under the curve BSA: Bovine serum albumine

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

Design/Setting/Participants: Twenty healthy trained men (averaging14,000 steps/d and engaged in sports) were randomly divided into a control group (no supplementation) and a cocktail group for a 20-day free-living intervention during which they stopped exercise and decreased their daily steps to averaging3000. During the last 10 days metabolic changes were further triggered by fructose overfeeding. On days 0, 10 and 20 body composition (DXA), blood chemistry, glucose tolerance (OGTT) and substrate oxidation (indirect calorimetry) were measured. Glucose tolerance included 1% fructose labelled with (U-13 C) fructose to assess liver de novo lipogenesis. Histological changes and related cellular markers were assessed from muscle biopsies collected on days 0 and 20.

Results: While the cocktail did not prevent the decrease in insulin sensitivity and its muscular correlates induced by the intervention, it fully prevented the hypertriglyceridemia, the drop in fasting HDL and total fat oxidation, and the increase in de novo lipogenesis. The cocktail further prevented the decrease in type-IIa muscle fiber cross-sectional area and was associated with lower protein ubiquitination content. The circulating anti-oxidant capacity was improved by the cocktail following the OGTT.

Conclusion:

A cocktail of nutrient compounds from dietary origin protects against the alterations in lipid metabolism induced by physical inactivity and fructose overfeeding.

INTRODUCTION

Over the past decade, physical inactivity has emerged as an important risk factor for a number of chronic metabolic and cardiovascular diseases. In addition, sedentary behaviors have been associated with adverse health effects [START_REF] Katzmarzyk | Bed rest and resistive vibration exercise unveil novel links between skeletal muscle mitochondrial function and insulin resistance[END_REF] even in subjects who meet the levels of current recommendations on physical exercise [START_REF] Bouchard | Less Sitting, More Physical Activity, or Higher Fitness?[END_REF]. This sedentary death syndrome [START_REF] Lees | Sedentary death syndrome[END_REF] is responsible for an equivalent number of deaths to smoking (19) and has prompted numerous research studies to understand the role of physical inactivity and sedentary behaviors in diseases aetiology and develop efficient preventive strategies.

Research on the physiology of physical inactivity in humans has been pioneered by researchers investigating physiological adaptations of astronauts to space environment. To do so, they used a ground-based model analogue to microgravity, the bed-rest model [START_REF] Hargens | Long-duration bed rest as an analog to microgravity[END_REF]. Indeed, the hypokinesia and hypodynamia induced during prolonged bed-rest are largely responsible for the described adaptations to space. Over the past 60 years, bed-rest studies ranging from as little as 3 days to 120 days have demonstrated that physical inactivity leads to muscle atrophy and a shift from slow oxidative fibers towards fast glycolytic fibers [START_REF] Narici | Disuse of the musculo-skeletal system in space and on earth[END_REF]. These structural adaptations are strongly associated with the development of metabolic inflexibility, low-grade inflammation and oxidative stress [START_REF] Mutin-Carnino | Effect of muscle unloading, reloading and exercise on inflammation during a head-down bed rest[END_REF][START_REF] Powers | Redox control of skeletal muscle atrophy[END_REF][START_REF] Rudwill | Metabolic Inflexibility Is an Early Marker of Bed-Rest-Induced Glucose Intolerance Even When Fat Mass Is Stable[END_REF].

There is an increase in fasting and post-prandial glucose oxidation concomitant to an increased spillover of dietary lipids along with a reduced capacity to burn fat (see (9) for review). The resultant hypertriglyceridemia leads to ectopic fat storage in the liver, muscle and bone marrow [START_REF] Trudel | A nutrient cocktail prevents lipid metabolism alterations 34 activities were resumed along with hemopoietic stimulation: the Women International Space Simulation for Exploration study[END_REF] and contributes to the development of insulin resistance at the muscle level (9). The role of physical inactivity and sedentary behaviors in the onset and progression of metabolic diseases have attracted an increasing attention from the scientific biomedical community over the past decade. Some investigators have proposed to reduce the number of daily steps of physically active individuals to 8 study the direct metabolic effects of physical inactivity [START_REF] Knudsen | Changes in insulin sensitivity precede changes in body composition during 14 days of step reduction combined with overfeeding in healthy young men[END_REF][START_REF] Krogh-Madsen | A 2-wk reduction of ambulatory activity attenuates peripheral insulin sensitivity[END_REF][START_REF] Olsen | Metabolic responses to reduced daily steps in healthy nonexercising men[END_REF]. While these studies confirmed the findings observed during the bed rest studies at levels of physical inactivity closer to what is observed in the general population, they did not examine the underlying mechanisms. The physiology of physical inactivity therefore needs to be further delineated in order to develop strategies to prevent its deleterious effects.

Recent studies conducted to develop preventive strategies suggest that bioactive nutrients such as polyphenols, vitamins and essential fatty acids may mitigate some metabolic features of physical inactivity. However so far, most studies have been tested in rodent analogs of microgravity to induce muscle disuse atrophy and metabolic disorders. A large body of data exists in human, rodents and primates demonstrating the effect of polyphenols such as quercetin, resveratrol, cinnamon, grape or green tea extracts on insulin sensitivity, lipid metabolism, inflammation and oxidative stress (see [START_REF] Alkhalidy | Dietary Flavonoids in the Prevention of T2D: An Overview[END_REF][START_REF] Guasch-Ferre | Dietary Polyphenols, Mediterranean Diet, Prediabetes, and Type 2 Diabetes: A Narrative Review of the Evidence[END_REF][START_REF] Hussain | Oxidative Stress and Inflammation: What Polyphenols Can Do for Us?[END_REF][START_REF] Mao | Oxidative stress-induced diseases and tea polyphenols[END_REF][START_REF] Murillo | The Relevance of Dietary Polyphenols in Cardiovascular Protection[END_REF][START_REF] Sosnowska | The role of nutraceuticals in the prevention of cardiovascular disease[END_REF][START_REF] Stull | Blueberries' Impact on Insulin Resistance and Glucose Intolerance[END_REF][START_REF] Woerdeman | Do grape polyphenols improve metabolic syndrome components? A systematic review[END_REF] for recent reviews). In addition, polyphenols supplemented as pure molecules such as 8-phenylnaringenin (flavanones, (53)), quercitin, (flavonols, (54)), resveratrol (oligostilbens, (51)), epigallocatechin 3 (catechins, (46)) or as extracts from dietary sources (green tea, apple extracts or grape seed extracts) prevent several aspects of rodent muscle atrophy and metabolic disorders induced by disuse while some promote a fatigue resistant muscle fiber phenotype [START_REF] Alway | Green tea extract attenuates muscle loss and improves muscle function during disuse, but fails to improve muscle recovery following unloading in aged rats[END_REF][START_REF] Lambert | Grape polyphenols supplementation reduces muscle atrophy in a mouse model of chronic inflammation[END_REF]46,[START_REF] Mizunoya | Fast-to-slow shift of muscle fiber-type composition by dietary apple polyphenols in rats: Impact of the low-dose supplementation[END_REF][START_REF] Onishi | Green tea extracts ameliorate high-fat diet-induced muscle atrophy in senescence-accelerated mouse prone-8 mice[END_REF].

Vitamin E is also known to have anti-oxidant capabilities, by acting as a reactive oxygen species (ROS) scavenger, and anti-inflammatory properties through inhibition of the NF-kB pathway which is well described in disuse atrophy models to activate proteolytic pathways [START_REF] Huey | In vivo vitamin E administration attenuates interleukin-6 and interleukin-1beta responses to an acute inflammatory insult in mouse skeletal and cardiac muscle[END_REF][START_REF] Khor | Vitamin E in sarcopenia: current evidences on its role in prevention and treatment[END_REF]. Vitamin E supplementation mitigates disuse atrophy in rats [START_REF] Servais | Dietary omega-3 fatty acid supplementation increases the rate of muscle protein synthesis in older adults: a randomized controlled trial[END_REF], reduces adipose tissue fibrosis, inflammation, oxidative stress and blood lipids in obese humans [START_REF] Alcala | Vitamin E reduces adipose tissue fibrosis, inflammation, and oxidative stress and improves metabolic profile in obesity[END_REF]. Vitamin E intake is also related to muscle strength in the elderly [START_REF] Ble | Lower plasma vitamin E levels are associated with the frailty syndrome: the InCHIANTI study[END_REF][START_REF] Cesari | Antioxidants and physical performance in elderly persons: the Invecchiare in Chianti (InCHIANTI) study[END_REF]. Selenium is often co-supplemented with vitamin E as it scavenges 9 ROS and boosts the intracellular effects of vitamin E. The combination of these two micronutrients was also shown to be beneficial on specific dystrophy [START_REF] Passerieux | Effects of vitamin C, vitamin E, zinc gluconate, and selenomethionine supplementation on muscle function and oxidative stress biomarkers in patients with facioscapulohumeral dystrophy: a double-blind randomized controlled clinical trial[END_REF]. Furthermore, selenium has independent effects on insulin sensitivity and reduces insulin secretion [START_REF] Tabrizi | The Effects of Selenium Supplementation on Glucose Metabolism and Lipid Profiles Among Patients with Metabolic Diseases: A Systematic Review and Meta-Analysis of Randomized Controlled Trials[END_REF]. Although these effects are still debated there are clear anti-oxidant and anti-inflammatory effects of selenium [START_REF] Farrokhian | Selenium Supplementation Affects Insulin Resistance and Serum hs-CRP in Patients with Type 2 Diabetes and Coronary Heart Disease[END_REF], but not on lipid metabolism [START_REF] Tabrizi | The Effects of Selenium Supplementation on Glucose Metabolism and Lipid Profiles Among Patients with Metabolic Diseases: A Systematic Review and Meta-Analysis of Randomized Controlled Trials[END_REF].

The role of omega 3 fatty acids (-3) has been studied for decades. Studies in healthy and unhealthy humans and animals showed that they improve hepatic insulin sensitivity, lower very low density lipoproteins (VLDL) production and de novo lipogenesis by the liver, and reduce inflammation and oxidative stress [START_REF] Stella | Update on the Impact of Omega 3 Fatty Acids on Inflammation, Insulin Resistance and Sarcopenia: A Review[END_REF][START_REF] Hodson | Docosahexaenoic acid enrichment in NAFLD is associated with improvements in hepatic metabolism and hepatic insulin sensitivity: a pilot study[END_REF][START_REF] Ng | Atorvastatin plus omega-3 fatty acid ethyl ester decreases very-low-density lipoprotein triglyceride production in insulin resistant obese men[END_REF]. All of these are metabolic features observed in the inactive/sedentary states (9). These observations were also reported in presence of fructose overfeeding [START_REF] Faeh | Effect of fructose overfeeding and fish oil administration on hepatic de novo lipogenesis and insulin sensitivity in healthy men[END_REF]. Surprisingly, besides the protective effects of -3 supplementation during bed-rest and spaceflight on bone markers (88), no studies have investigated their impact on muscle function during disuse. However, some studies suggest they could have beneficial effects to prevent against muscle atrophy.

In rodent cancer-induced cachexia, -3 fatty acids supplementation prevented muscle atrophy [START_REF] Smith | Effect of eicosapentaenoic acid, protein and amino acids on protein synthesis and degradation in skeletal muscle of cachectic mice[END_REF] and in the elderly, they increased activation of the protein synthesis mTOR pathway in response to insulin stimulation in the elderly (72, 73).

While the available literature suggests that some bio-active molecules, taken individually or as food extracts, improve several aspects of muscle and whole-body metabolic control, it is unlikely that any single micronutrient will be sufficiently powerful to reverse the wide range of deleterious effects induced by physical inactivity. Recently the notion of nutrient cocktails, to trigger additive and/or synergistic effects between bio-active compounds, has been proposed [START_REF] Bakker | An antiinflammatory dietary mix modulates inflammation and oxidative and metabolic stress in overweight men: a nutrigenomics approach[END_REF]. Several studies have shown that co-supplementing with -3 & green tea extracts (49), -3 & vitamin E [START_REF] Ebrahimi | The Effects of Omega-3 Fatty Acids and Vitamin E Co-Supplementation on Indices of Insulin Resistance and Hormonal Parameters in Patients with Polycystic Ovary Syndrome: A Randomized, Double-Blind, Placebo-Controlled Trial[END_REF][START_REF] Udupa | Study of comparative effects of antioxidants on insulin sensitivity in type 2 diabetes mellitus[END_REF], vitamin E & selenium [START_REF] Guertin | Effect of long-term vitamin E and selenium supplementation on urine F2isoprostanes, a biomarker of oxidative stress[END_REF], fish oil & selenium [START_REF] Wang | Skeletal muscle atrophy is attenuated in tumorbearing mice under chemotherapy by treatment with fish oil and selenium[END_REF], epigallocatechin & resveratrol (52) improve, to some 10 extent, insulin sensitivity, body composition, fat oxidation, inflammation and oxidative stress in aging and diseases associated with muscle atrophy including obesity and type 2 diabetes.

While these cocktails have the potential to reduce the risk of metabolic disease there are no studies in the literature examining their impact on physical inactivity-induced metabolic alterations in humans.

The purpose of this study was to determine the preventive effect of a nutrient cocktail composed of polyphenols, -3, vitamin E and selenium on the metabolic and physiological changes that occur during 20 days of reduced daily steps and exercise in trained men. To further trigger metabolic deterioration, dietary fructose supplementation was provided during the last 10 days of the trial. We hypothesized that the cocktail supplementation would reduce and/or prevent the deterioration in glucose and lipid metabolism, insulin sensitivity and muscle atrophy that are associated with reduced physical activity and fructose overfeeding.

METHODS

Subjects

Twenty healthy trained young men were recruited from the local community. Characteristics are presented in Table 1. Habitual physical activity was assessed using hip-worn triaxial accelerometry (Actigraph GT3x+ TM , Actigraph USA) for 7 days. Subjects were included if they walk >10,000 steps/day, participated in at least two leisure sport sessions per week and were free of any known diseases. Subjects were excluded if not able to drastically reduce professional, leisure and transportrelated physical activities. This study was approved by the local ethical committee and all subjects provided written informed consent (NCT03313869, ID-RCB number: 2015-A00665-14).

Experimental design 11

The experiment outflow is presented in Figure 1. Participants were randomly assigned to control (n=10) or daily nutrient cocktail supplemented (n=10) groups. Random tables were generated by the study statistician. The participants, nurses and physicians were not blinded as no placebo pills were given to the control group; however, the statistician was blinded. During the 20-day study in freeliving conditions, volunteers from both groups were asked to stop exercising and drastically reduce their daily physical activity. Subjects were instructed to walk a maximum of 2,500-4,000 steps/day. This was controlled on a daily basis by the subject and the investigators through a wrist-worn Fitbit pedometer (Fitbit, USA). Accurate data were assessed using an Actigraph GT3x+ TM hip-worn throughout the twenty days of the experiment. Metabolic stress induced by physical inactivity was boosted during the last ten days of the protocol by ingesting 3g/kg/d of fructose (Vivis® fructose) mixed with 0.5g/kg/d glucose in water to alleviate intestinal problems [START_REF] Ngo Sock | Effects of a short-term overfeeding with fructose or glucose in healthy young males[END_REF][START_REF] Tappy | Fructose and metabolic diseases: new findings, new questions[END_REF], on top of their regular diet. Fasting blood collection, glucose tolerance, substrate oxidation and de novo lipogenesis were measured at baseline and after 10 and 20 days of reduced activity. Muscle biopsies and body composition measurement were completed at baseline and after 20 days (Figure 1). Diet was not controlled during the intervention; subjects were instructed to eat according to their appetite.

Cocktail composition and doses

The supplemented group received a polyphenol nutrient cocktail derived from food sources that consist of Liliaceae, Vernenaceae, Lamiaceae, Vitaceae, Rubiaceae, Theaceae and Rutaceae Genres consisting of Allium cepa, Lippia citriodora, Ajuga reptans, Vitis vinifera, Coffea robusta, Camellia sinensis, and Citrus aurantium. The cocktail is referred to as XXS-2A and was designed by Spiral Company (Dijon, France). The daily dose was achieved by the ingestion of 3 pills (one at breakfast, lunch and dinner) to reach a total dose of 529.5 mg/d of polyphenols that was composed of 120 mg/d flavonols (including quercetin 50 mg), 75 mg/d oligostilbens (including resveratrol 20 mg), 91.5 mg/d hydroxycinnamic acids (including chlorogenic acid 40 mg), 135 mg/d flavanols (including 12 epigallocatechin gallate 60 mg) and 108 mg/d flavanones (including naringin 30 mg). As there is no Dietary References Intake (DRI) available for polyphenols, the ~500 mg/d dose was based on several reviews on the bioavailability and bioefficacy of polyphenols in humans and others studies that tested the effects of polyphenols on exercise performance and oxidative stress [START_REF] Manach | Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies[END_REF][START_REF] Myburgh | Polyphenol supplementation: benefits for exercise performance or oxidative stress?[END_REF][START_REF] Somerville | Polyphenols and Performance: A Systematic Review and Meta-Analysis[END_REF]. The 3 g daily dose of -3 (Omacor, Pierre Fabre Laboratories, Toulouse France) was based on French pharmacopeia recommendations for hypolipemic effects (2-4 g/day) and was provided as 1 pill per meal which is within the daily dose used in most clinical studies [START_REF] Hodson | Docosahexaenoic acid enrichment in NAFLD is associated with improvements in hepatic metabolism and hepatic insulin sensitivity: a pilot study[END_REF][START_REF] Ng | Atorvastatin plus omega-3 fatty acid ethyl ester decreases very-low-density lipoprotein triglyceride production in insulin resistant obese men[END_REF]. This daily dose thus corresponded to 1.1 g of eicosapentaenoic acid (EPA) and 1 g of docosahexaenoic acid (DHA).

Vitamin E and selenium were given as a single daily pill providing 168 mg of vitamin E associated with 80 µg of selenium (Solgar, Marne la Vallée, France). The DRI for Vitamin E is set at 15 mg/d.

The tolerable upper limit (UL) for intake is set at 1 g in adults, therefore the dose from the commercially available pill was 6 times lower than the UL but 11 times higher than DRI. To capitalize on the cocktail effect, the dose of vitamin E was two-fold lower the doses that have provided positive effects on metabolism and muscle [START_REF] Guertin | Effect of long-term vitamin E and selenium supplementation on urine F2isoprostanes, a biomarker of oxidative stress[END_REF][START_REF] Passerieux | Effects of vitamin C, vitamin E, zinc gluconate, and selenomethionine supplementation on muscle function and oxidative stress biomarkers in patients with facioscapulohumeral dystrophy: a double-blind randomized controlled clinical trial[END_REF]. Regarding selenium, the intake reported in most countries presents a large variability. DRI and UL are respectively set at 55 µg and 400 µg per day. The selected dose provided a daily supplement that set the daily intake at 135 µg/d which is 3 times lower than the UL and lower than the dose reported in previous studies [START_REF] Guertin | Effect of long-term vitamin E and selenium supplementation on urine F2isoprostanes, a biomarker of oxidative stress[END_REF][START_REF] Passerieux | Effects of vitamin C, vitamin E, zinc gluconate, and selenomethionine supplementation on muscle function and oxidative stress biomarkers in patients with facioscapulohumeral dystrophy: a double-blind randomized controlled clinical trial[END_REF] in order to capitalise on cocktail effects. The control group did not receive any supplementation or placebo.

Body composition

Fat mass (FM) and lean body mass (LBM) were assessed by using a dual-energy X-ray absorptiometer (DXA, HOLOGIC QDR 4500W, USA) at baseline and on day 20 of the experiment.

Physical Activity Patterns

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Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations 13 Time spent in sitting and active was determined using a tri-axial accelerometer (ActiGraph GT3X+; ActiGraph, Pensacola, Fla., USA). Participants were instructed to wear the accelerometer at their right hip at all times except for bathing during one week before the inclusion and during the all experimental protocol. At each visit, both raw accelerometry and activity-counts per min were downloaded using manufacturer software (Actilife 6.13, Pensacola, USA). An automatic activityrecognition algorithm [START_REF] Bastian | Automatic identification of physical activity types and sedentary behaviors from triaxial accelerometer: laboratory-based calibrations are not enough[END_REF], that identifies sitting time, and an activity-specific energy expenditure model [START_REF] Garnotel | Prior automatic posture and activity identification improves physical activity energy expenditure prediction from hip-worn triaxial accelerometry[END_REF], both developed by our group, were used to determine time spent in a sitting position and in different activity intensities; cut-points of 1.5-3 METs and >3METs were used for light intensity activity and moderate-to-very vigorous activity, respectively.

Glucose tolerance and substrate use

Subjects reported to the clinic the evening prior to the test. A standard dinner was given containing 47.7% carbohydrates, 32.5% lipids and 17.8% protein for a total of 918 Kcal intake. An oral glucose tolerance test (OGTT) was performed after an overnight fast using a mix of 1g/kg of glucose and 0.5g/kg of fructose diluted in 300mL of water; 1% of fructose was labelled with U- 13 C-fructose (Eurisotop, Paris). Following baseline collection and glucose/fructose ingestion, blood samples were collected every 15-min for the first 3 hours and every 30-min for the 4 th hour. Carbohydrate and fat oxidation rates were determined every hour using canopy dilution respirometry (Quark, Cosmed, Italy) and the classical equations of indirect calorimetry corrected for urinary nitrogen excretion.

Insulin sensitivity was estimated using the Matsuda Index [START_REF] Matsuda | The Green Tea Polyphenol Epigallocatechin-3-Gallate (EGCg) Attenuates Skeletal Muscle Atrophy in a Rat Model of Sarcopenia[END_REF]. Metabolic flexibility, defined as the ability to adjust nutrient oxidation to nutrient availability and demand, was assessed during the OGTT by examining the relationship between the variance of plasma insulin and the variance of non-protein respiratory quotient (NPRQ). As explained previously [START_REF] Bergouignan | Breast cancer affects both the hippocampus volume and the episodic autobiographical memory retrieval[END_REF] the variance-derived indexes assume a metabolically flexible state when the variance in insulin is low and the variance in NPRQ is 14 high; in other words, when the body has a high capacity to switch from fat to carbohydrate oxidation in association with small changes in insulin concentration in response to the OGTT. A transition towards a metabolically inflexible state is assumed when the variance in insulin increases and/or the variance in NPRQ decreases.

Exogenous fructose oxidation

Exhaled breath samples were collected at the same time as blood samples. Breath 13 C/ 12 C isotopic ratio was measured in triplicate on a GasBench system (Thermo Scientific, Germany) connected to a continuous-flow isotope ratio mass spectrometer (IRMS, DELTA V, Thermo Scientific, Germany). U-13 C-fructose oxidation was calculated as the cumulated percentage dose recovery of 13 C in expired CO 2 per hour over the OGTT, as previously described [START_REF] Lefai | Exercise training improves fat metabolism independent of total energy expenditure in sedentary overweight men, but does not restore lean metabolic phenotype[END_REF].

Liver de novo lipogenesis

De novo lipogenesis was measured as the apparition of 1-13 C-palmitate from U- 13 C-fructose in very low-density lipoprotein (VLDL). In brief, as previously described (42) VLDL were separated by sequential ultracentrifugation and total lipids were extracted from VLDL by a modified Folch technique. TGs were further separated by solid phase extraction and derivatized into methyl esters.

The absolute concentration of both unlabelled and labelled palmitate was measured by gas chromatography/mass spectrometry (Agilent 5975, Inert XL) through a dual acquisition program in single ion monitoring m/z ratios of 270 and 271, and calculated by reference to internal standards added to the plasma. The concentration of 1-13 C-palmitate was calculated by multiplying its molar percent enrichment (MPE) by the concentration of total palmitate. Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations 15 Plasma insulin was assessed by radio-immuno assay, and glucose, NEFA and TG were measured by colorimetric assays and enzymatic methods, as previously described [START_REF] Lefai | Exercise training improves fat metabolism independent of total energy expenditure in sedentary overweight men, but does not restore lean metabolic phenotype[END_REF]. Reduced and oxidized glutathione (GSH and GSSG, respectively) concentrations were simultaneously measured by reverse-phase high performance liquid chromatography (HPLC) as previously described [START_REF] Yilmaz | A Practical HPLC Method to Measure Reduced (GSH) and Oxidized (GSSG) Glutathione Concentrations in Animal Tissues[END_REF]. Total plasma malondialdehyde (MDA) was determined by reverse-phase HPLC [START_REF] Tüközkan | Measurement of Total Malondialdehyde in Plasma and Tissues by High-Performance Liquid Chromatography and Thiobarbituric Acid Assay[END_REF]. The antioxidant defenses were examined using a test based on in vitro free-radical-induced blood hemolysis KRL test (Kirial International/Spiral, Couternon, France) as previously described [START_REF] Rifler | A moderate red wine intake improves blood lipid parameters and erythrocytes membrane fluidity in post myocardial infarct patients[END_REF].

Blood sample analyses

Muscle biopsies and parameters

Muscle biopsies were performed on the mid Vastus Lateralis (VL) under aseptic conditions and after anaesthesia (2% w/v lidocaine HCl) using Bergström skeletal muscle biopsy needle (Lauran Médical®, KBM 5/10). One piece was mounted in tragacanthum gum (OCT Compound) for histological analysis, cooled to the temperature of liquid nitrogen in isopentane, and the remainder was snap frozen in liquid nitrogen and stored at -80°C until further analysis.

Immunohistochemical classification of muscle fibers and cross-sectional area measurements:

Transverse serial cross sections (10 m thick) of vastus lateralis muscle samples were obtained using a cryostat at -25°C (HM-560, Microm H), and mounted onto glass microscope slides. Before labeling, sections were dried and fixed for 10 min in acetone. Sections were then washed in phosphate buffered saline (PBS), blocked and permeabilized with 0.1% Triton-X100 and 20% horse serum. For muscle fiber typing and cross sectional areas (CSA) determination, sections were incubated with anti-MyHC primary antibodies (anti-slow (I) MyHC, BA-D5, Developmental Studies Hybridoma Bank, 1:10; anti-fast (II) MyHC, M4276, Sigma-Aldrich, 1:200), and anti-fast (IIA) MyHC, SC-71, Developmental Studies Hybridoma Bank, 1:10) for 1 h at 37°C, followed by washes in PBS and incubation with the secondary antibodies (ALEXA 488, A11029, Invitrogen, 1:800; 16 ALEXA 568, A11031, Invitrogen, 1:800) for 1 h. Fiber typing was manually determined, and the fiber sizes were analyzed with Image J software (1.46r version). A total of approximately 300 myofibers per sample were thus measured in double-immunostained pre-and post-DI VL cryosections. Citrate Synthase Activity: Citrate synthase activity of muscle samples was measured using a commercially available kit, as reported before (36).

Protein expression:

Data and statistical analysis

The effects of the intervention and the cocktail supplementation on the outcomes were assessed by using mixed linear models with group, intervention, group-by-intervention interaction and baseline values as fixed effects and subjects as random effect. Additional adjustment for changes in FM and LBM was performed for substrate oxidation. Between-group and within-group differences were assessed by using post-hoc tests. Data obtained during OGTT are presented as area under the curve (AUC). Significance was set up at 0.05 for main effects and 0.10 for interaction effects. Values are mean ± SEM, unless otherwise stated. Statistical analysis were performed with SAS version 9.4 (SAS Institute, Cary, USA).

RESULTS

Subjects' characteristics and changes in time spent physically active and sitting

Subjects' characteristics and changes in activities are presented in Table 1. Participants' compliance was very good; over the 20-day intervention daily steps went down from 14,952 ± 1,720 steps to 3,009 ± 298 steps in the supplemented group and from 13,032 ± 875 steps to 2,645 ± 331 steps in the control group. We observed in both groups a reduction of both light-intensity activity and moderateto-very vigorous intensity activity, along with a significant increase in time spent sitting during waking hours. Although the intervention did not modify body and lean mass, we observed a significant increase in FM, of less than 1kg, independent of the cocktail supplementation.

Glucose tolerance and insulin sensitivity

Fasting plasma glucose and insulin were neither affected by the intervention nor by the cocktail supplementation (Table 2). Concentrations of glucose, TG and insulin during the OGTT are presented in Figures 2A,2B, 2C respectively. To increase readability of the results, data are also presented as AUC in Figure 3. During the OGTT, glucose concentration did not change between control and supplemented groups (Figure 3B); insulin increased after 10 days of reduction in daily steps (Figure 3A) but did not further change after 10 more days of inactivity combined with fructose

Version postprint

Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations 18 overfeeding. Reduction in daily steps decreased insulin sensitivity in both groups as indicated by the decrease in Matsuda index (Figure 3C). Fasting high molecular weight (HMW) adiponectin increased during the intervention in both groups, but the increase was greater in the supplemented group compared to the control group after 20 days of intervention (Table 2). Total carbohydrate oxidation (Figure 3D) and exogenous fructose oxidation (Figure 3E) during the OGTT increased similarly in both groups after both 10 and 20 days of intervention. While a group-by-intervention interaction almost reached significance for an increase in insulin variance, significant decrease in NPRQ variance was observed during the OGTT in the control group after both 10 and 20 days of intervention, that was fully prevented by the supplementation (Figure 3F). This indicates a development of metabolic inflexibility in the control group but not in the supplemented group, that was likely due to decreased oxidative capacities in association with whole body metabolic adaptations. Of note, most metabolic changes were observed after 10 days of inactivity only; the addition of 10 more days of reduced daily steps combined with fructose overfeeding did not lead to further modifications in metabolic flexibility.

Lipid metabolism

Fasting TG and HDL respectively increased and decreased in the control group after both 10 and 20 days of intervention (Table 2). Similar responses were observed during the OGTT. TG (Figure 4A), VLDL-TG (Figure 4B), palmitate VLDL-TG (Figure 4C) and de novo lipogenesis from fructose (Figure 4D) gradually and significantly increased after 10 and 20 days of intervention in the control group. The intervention decreased total lipid oxidation in association with an increase in net lipid synthesis both in the fasting states and following the OGTT (Table 4 & Figure 4E), as indicated by the negative values of lipid oxidation, thus confirming the increase in de novo lipogenesis as measured by stable isotopes. The cocktail supplementation fully prevented the increase in lipemia (Table 2) in both fasting state and during the OGTT (Figure 4A), the decrease in total lipid Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (147.100.066.219) on October 8, 2018.

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Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations 19 oxidation (Table 2 and Figure 4E) and the increase in de novo lipogenesis measured both by tracer technique (Figure 4D) and indirect calorimetry (Table 2) induced by the daily step reduction with or without fructose overfeeding.

Anti-oxidant capacity

Fasting oxidative markers remained unaffected by either the intervention or the supplementation (Table 3). However, blood anti-oxidant capacity following the OGTT decreased in the control group when the fructose overfeeding was added to the daily step reduction while the supplemented group remained at significantly higher values of whole blood anti-oxidant capacity (Figure 3F).

Skeletal muscle analysis

Muscle parameters are presented in Table 5. While type I fibers CSA was lower in both groups at the end of the intervention, the decrease in type IIa CSA observed in the control group, was prevented by the cocktail supplementation. In line with this observation, ubiquitination content, a protein marker of proteolysis processes, increased only in the control group. At the mitochondrial level both Mitofusin-2 and PGC1α contents dropped in response to the intervention. We failed to see a protective effect of the cocktail. FATP1, involved in fatty acid transportation at the mitochondrial level, tended to rise in the cocktail group only (p=0.07), supporting the maintenance of lipid oxidation during the intervention in the supplemented but not in the control group. No changes in citrate synthase activity, skeletal muscle intracellular signaling protein contents and oxidative stress markers were observed.

DISCUSSION

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Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations 20

The main findings of this randomized interventional study in lean healthy trained young male are that (i) a reduction in daily light intensity activity and moderate-to-vigorous activity along with an increase in time spent sedentary leads to decreased insulin sensitivity, total fat oxidation, hyperlipemia and increased de novo lipogenesis; and (ii) a nutrient cocktail supplement prevents the alterations on lipid metabolism but not insulin sensitivity. This is the first study to test the efficacy of a nutrient cocktail to counteract the negative effects of physical inactivity along with fructose overfeeding.

During the intervention, subjects decreased on average their activity to <3,000 steps per day. Body weight and LBM remained stable during the study but FM and the percentage of body fat increased.

The effect on body weight is not surprising. It is likely to be influenced by 1) the known delayed compensatory adjustment in energy intake, 2) the duration of the intervention, given some (27,[START_REF] Knudsen | Changes in insulin sensitivity precede changes in body composition during 14 days of step reduction combined with overfeeding in healthy young men[END_REF], but not all studies (5, 26) with 7-14 days decreased activity reported weight gain, and 3) the fructose overfeeding between 10-20 days, as others have also reported an increase in FM with overfeeding [START_REF] Knudsen | Changes in insulin sensitivity precede changes in body composition during 14 days of step reduction combined with overfeeding in healthy young men[END_REF]. The increase in FM is thus in line with the design of the study. Overall, LBM was preserved during the study though there was a decrease in type II muscle fibers CSA in the control group, suggesting that the cocktail supplementation may delay muscle disuse atrophy.

There was a decrease in insulin sensitivity during the 20-day intervention, in agreement with other studies when activity was reduced for 3-14 days [START_REF] Arciero | Effects of short-term inactivity on glucose tolerance, energy expenditure, and blood flow in trained subjects[END_REF]27,[START_REF] Holwerda | The influence of reduced insulin sensitivity via short-term reductions in physical activity on cardiac baroreflex sensitivity during acute hyperglycemia[END_REF][START_REF] Knudsen | Changes in insulin sensitivity precede changes in body composition during 14 days of step reduction combined with overfeeding in healthy young men[END_REF][START_REF] Mikus | Lowering physical activity impairs glycemic control in healthy volunteers[END_REF][START_REF] Reynolds | Acute inactivity impairs glycemic control but not blood flow to glucose ingestion[END_REF]. This was mainly due to increased insulin AUC during the OGTT while we did not show a change in glucose, in agreement with some (2,9) but not all studies [START_REF] Arciero | Effects of short-term inactivity on glucose tolerance, energy expenditure, and blood flow in trained subjects[END_REF][START_REF] Holwerda | The influence of reduced insulin sensitivity via short-term reductions in physical activity on cardiac baroreflex sensitivity during acute hyperglycemia[END_REF]. The additional fructose provided after day 10 did not affect insulin sensitivity but there was a significant increase in carbohydrate oxidation and exogenous fructose oxidation. Knudsen et al. [START_REF] Knudsen | Changes in insulin sensitivity precede changes in body composition during 14 days of step reduction combined with overfeeding in healthy young men[END_REF] reported similar glucose and insulin responses to an OGTT following 14 days of reduced activity and a 50% increase in energy intake. While the

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Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations [START_REF] Faeh | Effect of fructose overfeeding and fish oil administration on hepatic de novo lipogenesis and insulin sensitivity in healthy men[END_REF] Matsuda index, in that study, was significantly lower at day 7 but not day 14, clamp-derived insulin sensitivity had decreased by ~44%. In agreement with other studies of decreased activity [START_REF] Knudsen | Changes in insulin sensitivity precede changes in body composition during 14 days of step reduction combined with overfeeding in healthy young men[END_REF] or bedrest [START_REF] Jurdana | Impact of 14day bed rest on serum adipokines and low-grade inflammation in younger and older adults[END_REF], we reported an increase in fasting plasma adiponectin after 20 days of decreased activity. This is in contrast with studies where decreased adiponectin is associated with insulin resistance. It is possible that, in these healthy individuals, an increase in adiponectin may be a short-term compensatory mechanism to preserve insulin sensitivity. In support of this we did not find a change in Akt and GSK3 proteins following the intervention. This would need to be further investigated but given the preservation of metabolic flexibility, the effects appear to be more pronounced on lipid metabolism.

By using bed-rest ranging from 7 to 90 days in men and women we have shown that a hallmark of physical inactivity physiology is a decrease in exogenous lipid clearance due to both a reduced uptake and oxidative capacity at the muscle level (see [START_REF] Bergouignan | Breast cancer affects both the hippocampus volume and the episodic autobiographical memory retrieval[END_REF] for review). We further showed in the general population through training/detraining studies that activity energy expenditure is a major determinant of lipid trafficking (plasma pools, chylomicrons, VLDL) and partitioning through uptake (LPL, FAT/CD36, FABPpm) between oxidation (LCcoA synthase, CPT1) and storage (mtGPAT) within the skeletal muscle [START_REF] Bergouignan | Activity energy expenditure is a major determinant of dietary fat oxidation and trafficking, but the deleterious effect of detraining is more marked than the beneficial effect of training at current recommendations[END_REF]. In the present study, we observed that physical inactivity by voluntary step reduction and physical inactivity plus fructose overfeeding gradually decreased fasting and postprandial fat oxidation to an extent where only net lipid synthesis was observed during the OGTT; similar to our observation during an OGTT following 7-days bed-rest in men and women [START_REF] Blanc | Fuel homeostasis during physical inactivity induced by bed rest[END_REF]. One of the striking results of the present study is the capacity of the cocktail to fully prevent the effects on lipid oxidation, thus likely preventing the hypertriglyceridemia of the control group.

Although no similar data exist, numerous recent studies investigating the impact of various bioactive compounds from the diet, taken as cocktails, in various physio-pathological states and models Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (147.100.066.219) on October 8, 2018.

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Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations 22 support our results [START_REF] Annuzzi | Diets naturally rich in polyphenols improve fasting and postprandial dyslipidemia and reduce oxidative stress: a randomized controlled trial[END_REF][START_REF] Bondia-Pons | Isoenergetic diets differing in their n-3 fatty acid and polyphenol content reflect different plasma and HDL-fraction lipidomic profiles in subjects at high cardiovascular risk[END_REF][START_REF] Hodson | Docosahexaenoic acid enrichment in NAFLD is associated with improvements in hepatic metabolism and hepatic insulin sensitivity: a pilot study[END_REF][START_REF] Most | Combined epigallocatechin-3-gallate and resveratrol supplementation for 12 wk increases mitochondrial capacity and fat oxidation, but not insulin sensitivity, in obese humans: a randomized controlled trial[END_REF]. However, mechanistic evidence primarily comes from rodent models. Vitamin E plus -3 fatty acids supplementation was hepato-protective in naturally aging rats, with major anti-oxidant properties, especially in the brain [START_REF] Narayanankutty | Vitamin E supplementation modulates the biological effects of omega-3 fatty acids in naturally aged rats[END_REF]. It was argued that vitamin E reduces the peroxidation of -3 fatty acids thus allowing synergistic effects. In support of that, fish oils associated with grape polyphenols in rats fed a high sucrose diet improved numerous blood biochemical parameters such as HDL and TG, as in our study, by significantly enhancing fatty acid beta oxidation [START_REF] Mendez | Changes in liver proteins of rats fed standard and high-fat and sucrose diets induced by fish omega-3 PUFAs and their combination with grape polyphenols according to quantitative proteomics[END_REF]. Interestingly, the same study showed a major suppression of lipogenic enzymes by their cocktail, in agreement with our human data. A recent review [START_REF] Rodriguez-Ramiro | Polyphenols and non-alcoholic fatty liver disease: impact and mechanisms[END_REF] reported evidence that polyphenols play key controlling roles in suppressing de novo lipogenesis in various models of nonalcoholic fatty liver diseases. Selenium is often co-supplemented with vitamin E but is likely to have had limited impact in this study. Supplementation with selenium has independent effects on insulin sensitivity and insulin secretion but no effect on lipid profiles [START_REF] Tabrizi | The Effects of Selenium Supplementation on Glucose Metabolism and Lipid Profiles Among Patients with Metabolic Diseases: A Systematic Review and Meta-Analysis of Randomized Controlled Trials[END_REF]. In this study the main changes were in lipid metabolism with no changes in glucose/insulin parameters.

The beneficial effects of fish oils -3 fatty acids on lipemia by lowering TG and rising HDL have been well known for decades [START_REF] Yetiv | Clinical applications of fish oils[END_REF]. In overweight/obese subjects supplemented with -3 fatty acids and polyphenols there is a substantial improvement in fasting plasma HDL and their sub-classes [START_REF] Bondia-Pons | Isoenergetic diets differing in their n-3 fatty acid and polyphenol content reflect different plasma and HDL-fraction lipidomic profiles in subjects at high cardiovascular risk[END_REF] as well as post-prandial TG and triglyceride-rich lipoproteins (4). Annuzzi et al. [START_REF] Annuzzi | Diets naturally rich in polyphenols improve fasting and postprandial dyslipidemia and reduce oxidative stress: a randomized controlled trial[END_REF] showed reduced oxidative damage through a reduced excretion of 8-isoprostane. This data supports the improved global anti-oxidant capacity we observed in our study during the OGTT. The impact on oxidative stress could have been augmented by the co-supplementation of vitamin E and selenium, both recognized as ROS scavengers [START_REF] Huey | In vivo vitamin E administration attenuates interleukin-6 and interleukin-1beta responses to an acute inflammatory insult in mouse skeletal and cardiac muscle[END_REF][START_REF] Khor | Vitamin E in sarcopenia: current evidences on its role in prevention and treatment[END_REF][START_REF] Passerieux | Effects of vitamin C, vitamin E, zinc gluconate, and selenomethionine supplementation on muscle function and oxidative stress biomarkers in patients with facioscapulohumeral dystrophy: a double-blind randomized controlled clinical trial[END_REF]. Of note, the impact of our cocktail on various markers of anti-oxidant capacity or oxidative markers was quite modest. One may thus argue that, in our study, the principal effects were due to the -3 fats, known to boost the whole muscle machinery of lipid metabolism [START_REF] Chorner | Dietary alphalinolenic acid supplementation alters skeletal muscle plasma membrane lipid composition, sarcolemmal FAT/CD36 abundance, and palmitate transport rates[END_REF], and to a lesser extent to the polyphenols and vitamin E/selenium. However, a

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Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations 23 recent study of 12-week supplementation with epigallocatechin-3-gallate and resveratrol showed major impact on mitochondrial capacity and fat oxidation in overweight/obese men and women [START_REF] Most | Combined epigallocatechin-3-gallate and resveratrol supplementation for 12 wk increases mitochondrial capacity and fat oxidation, but not insulin sensitivity, in obese humans: a randomized controlled trial[END_REF].

In agreement with our data, they did not observe an improvement in insulin sensitivity. In addition, Altogether these data support the positive effect on lipid metabolism associated with the cocktail supplementation in our study.

While there is strong evidence which support our results on lipid metabolism following polyphenols and -3 supplementation, we also observed interesting effects on the muscle phenotype itself. Indeed, the muscle is known to be plastic in response to mechanical loading and unloading and it was expected that reduction in physical activity, by ~10000 steps/d, would have a detraining and deconditioning effect in healthy trained subjects. There was a global decrease in muscle fiber CSA with significant reductions in type 1 and type 2 fibers. Such plasticity in response to exercise or chronic hypoactivity is well known [START_REF] Chopard | Molecular events and signalling pathways involved in skeletal muscle disuse-induced atrophy and the impact of countermeasures[END_REF]. Interestingly, these adverse effects on type 2 muscle fibers were prevented by the cocktail and may be associated with the decrease in ubiquitination content, factors known to be associated to muscle deconditioning and fiber atrophy. We have previously shown that resveratrol prevented disuse muscle atrophy of hind-limb suspended rats [START_REF] Momken | Resveratrol prevents the wasting disorders of mechanical unloading by acting as a physical exercise mimetic in the rat[END_REF] and a recent study reported that -3 fatty acids delays muscle degradation in mice [START_REF] Soni | Eicosapentaenoic and Docosahexaenoic Acid-Enriched High Fat Diet Delays Skeletal Muscle Degradation in Mice[END_REF]. It is possible that these components of the nutrient cocktail are responsible for preserving type 2 fiber CSA in this study as there is no evidence for a role of vitamin E or selenium. However, there are no mechanistic studies examining the effects of nutrient cocktails and further experiments are required.

24

Limitations must be acknowledged. The control group did not take a placebo supplement due to difficulties to find a neutral oil to encapsulate. This being said the magnitude of the effects we observed suggest this would have a minor impact on the conclusion of the study. The molecular parameters we selected did not allow us to fully unravel the mechanisms at play and further mechanistic studies are required. Finally, we acknowledge that subjects taking >14,000 step/d does not represent the general population and the interpretation of the results are delimited to young active men. While recommending physical activity is a central component of health promotion policies, not enough people achieve the recommendations and therefore nutritional strategies to offset some of the deleterious effects of physical inactivity are important. Therefore, further studies are needed on less active populations and in at-risk populations for developing metabolic diseases.

In conclusion, we have demonstrated that the negative effects of physical inactivity on lipid metabolism can be mitigated by micronutrient supplementation. We have established that the early decrements in lipid oxidation contribute to increased de novo lipogenesis but that a nutrient cocktail containing polyphenols, -3 fatty acids and other compounds can effectively prevent these changes during 20-days of decreased physical activity. These data highlight the importance of regular physical activity to maintain carbohydrate and lipid metabolism. It is possible that this supplementation could be an effective prevention strategy for chronic diseases such as cardiovascular disease and type 2 diabetes but long-term studies, especially in high risk groups, are now required. Copyright © 2018 American Physiological Society. All rights reserved.

Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations !"##$%

6 - 3 :

 63 density lipoprotein HMW: High molecular weight HPLC: High performance liquid chromatography IRMS: Isotope ratio mass spectrometry LBM: Lean body mass LCcoA: Long chain acyl co-A LPL: Lipoprotein lipase MDA: Malondialdehyde MET: Metabolic equivalent MPE: Molar percent enrichment mtGPAT: mitochondrial glycerol-3phosphate acyltransferase NEFA: Non-esterified fatty acids NFkB: Nuclear factor kappa beta NPRQ: non-protein respiratory quotient OGTT: Oral glucose tolerance test PBS: Phosphate buffered saline PGC1: Peroxysome proliferator-activated receptor gamma co-activator 1 alpha ROS: Reactive oxygen species TG: Triglycerides UL: Upper limit VL: Vastus lateralis VLDL: Very low-density lipoprotein Omega-3 fatty acids 122

  Muscle samples were homogenized in 10 volumes of lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 100 mM NaF, 5 mM Na3VO4, 1% Triton X-100, 1% SDS, 40 mM β-glycerophosphate and protease inhibitor mixture [P8340; Sigma-Aldrich]) and centrifuged at 10,000 g for 10 min (4°C). Sixty micrograms of protein extract were loaded into Stain-Free 4-20% precast gels (4568095; Bio-Rad) before electrophoretic transfer onto nitrocellulose membranes (Bio-Rad; Trans-Blot Turbo Blotting System). After transfer, the membranes were blocked with 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20 (TBS-T) containing 5% skimmed milk or bovine serum albumin (BSA) and incubated overnight at 4°C with primary antibodies. The membranes were then incubated for 1 h with a peroxidase-conjugated secondary antibody. The immunoblots were revealed using a Pierce ECL kit (32106; Thermo Scientific), and proteins were visualized by enhanced chemiluminescence using the ChemiDoc Touch Imaging System and quantified with Image Lab™ Touch Software (version 5.2.1). Stain-Free technology or red Ponceau or GAPDH were used as loading control.

  Annuzzi et al. (4) found that the polyphenols reduced circulating TG and VLDL while -3 reduced postprandial cholesterol and VLDL apolipoprotein B-48. While the cocktail may benefit physically inactive and unhealthy individuals, it could also optimize the effects of training on lipid metabolism. In this line, Ota et al. showed supplementation in green tea extract beverage rich in catechins for two months, concomitant to aerobic exercise training increased fat oxidation rates during exercise (64).

Figure 1 :Figure 2 :

 12 Figure 1: Design of the study

Figure 3 :

 3 Figure 3: Carbohydrate metabolism

Figure 4 :

 4 Figure 4: Lipid metabolism and free radicals defense

Table 1 :Table 2 :Table 4 :

 124 Anthropometry and physical activity dataMeans are ± SEM.1 Mixed models are adjusted on baseline. Fasting metabolites and hormones Means are ± SEM.1 Mixed models are adjusted on baseline.Post-hoc tests: *p<0.05, **p<0.01, ***p<0.001 vs control. #p<0.05, ##p<0.01, ###p<0.001 vs baseline. HOMA index: Homeostasis model assessment of insulin resistance; TG: Triglycerides; HDL: High Density Lipoprotein; LDL: Low Density Lipoprotein; HMW: High Molecular Weight Table 3: Fasting oxidative stress and damages Means are ± SEM. 1 Mixed models are adjusted on baseline. Post-hoc tests: *p<0.05, **p<0.01, ***p<0.001 vs control. KRL: Total antiradical defense potential (in French, Kit Radicaux Libres); Reseda (in French, RESErves Défenses Antioxydantes) 1, 2 and 3: antiradical defense reserves unmasked by treatment of serum with respectively glucosides, sulphates or glucuronides. GSH: Reduced Glutathione; GSSG: Oxidized Glutathione. Fasting energy & substrate oxidation 1 Mixed models are adjusted on baseline, fat mass and fat-free mass. Data are presented as LSMeans ± SEM. Post-hoc tests: *p<0.05, **p<0.01, ***p<0.001 vs control. RMR: Resting metabolic rate; NPRQ: Non-protein respiratory quotient Days AUC (mM/L * 240 min) in NPRQ over 240 min OGTT Variance in INSULIN over 240 min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www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (147.100.066.219) on October 8, 2018.

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Acknowledgements

We are thankful to the participants and the medical, nutrition and administrative staff of the MEDES -Institut de Médecine et Physiologie Spatiale.

Funding: This work was supported by grants from the CNRS and from the European (ESA) and French (CNES) Space Agencies. AD and RD are supported by graduate fellowships from CNES. Clinical trial registration: NCT03313869 Disclosure summary Michel Prost, director of Spiral company developed the cocktail XXS-2A to market it if efficient. Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (147.100.066.219) on October 8, 2018.

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Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations

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Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations Copyright © 2018 American Physiological Society. All rights reserved.

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Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations Copyright © 2018 American Physiological Society. All rights reserved.

Version postprint

Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations Copyright © 2018 American Physiological Society. All rights reserved.

Version postprint

Comment citer ce document : Damiot, A., Demangel, R., Noone , J., Chery, I., Zahariev, A., Normand, S., Brioche, T., Crampes, F., De Glisezinski, I., Lefai, E., Bareille, M.-P., Chopard, A., DRAI, J., Collin-Chavagnac, D., Heer, M., Gauquelin-Koch, G., Prost , M., Simon , P., Py, G., Blanc , S., Simon, C., Bergouignan, A., O'Gorman, D. (2019). A nutrient cocktail prevents lipid metabolism alterations Copyright © 2018 American Physiological Society. All rights reserved.


Jianluo

Yan Liu

Songqiu Yang

Amandine L Flourat

Florent Allais

Keli Han
email: klhan@dicp.ac.cn

Ultrafast Barrierless Photoisomerization and Strong UV Absorption of Photoproducts in Plant Sunscreens

Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083 2 ABSTRACT: Sunscreens are aimed at protecting skins from solar UV irradiation. By utilizing femtosecond transient absorption spectroscopy and time-dependent density functional theory, we explain nature's selection of sinapoyl malate rather than sinapic acid as the plant sunscreen molecule. In physiological pH condition, the two molecules are deprotonated and their excited ππ* states are found to relax to the ground states in a few tens of picoseconds via a barrierless trans-cis photoisomerization. After forming the cis-photoproduct, the efficacy of sinapic acid is reduced much. In contrast, the efficacy of sinapoyl malate is affected slightly because the cisproduct still absorbs UV light strongly. In addition, protonated sinapic acid is found to be a good potential sunscreen molecule. 3 An over exposure to ultraviolet (UV) radiation has deleterious effects on animals and higher plants, and with the depletion of the ozone layer due to the increasing pollution, this exposure of UV is increasing day by day. [START_REF] Gonzalez | The Latest on Skin Photoprotection[END_REF][START_REF] You | Cyclobutane Pyrimidine Dimers Are Responsible for the Vast Majority of Mutations Induced by UVB Irradiation in Mammalian Cells[END_REF][START_REF] Cadet | DNA Base Damage by Reactive Oxygen Species, Oxidizing Agents, and UV Radiation[END_REF] Various sunscreens are designed to protect skins from damages by UV light, such as oxybenzone [START_REF] Baker | Probing the Ultrafast Energy Dissipation Mechanism of the Sunscreen Oxybenzone after UVA Irradiation[END_REF] and cinnamate-based molecules. [START_REF] Tan | Excited-State Dynamics of Isolated and Microsolvated Cinnamate-Based UV-B Sunscreens[END_REF][START_REF] Miyazaki | Direct Spectroscopic Evidence of Photoisomerization in para-Methoxy Methylcinnamate Revealed by Low-Temperature Matrix-Isolation FTIR Spectroscopy[END_REF][START_REF] Chang | Photoprotection Mechanism of p-Methoxy Methylcinnamate: A CASPT2 Study[END_REF] A good sunscreen agent should have a large absorption section on UVB (285-320 nm) and UVA (320-400 nm) light and dissipate the energy into surroundings efficiently. [START_REF] Kockler | Photostability of Sunscreens[END_REF] Plants sustain much longer exposure to UV radiation than animals. As a result, nature has selected efficient screening agents to protect plants.

In adult Arabidopsis plants, sinapoyl malate is deposited in the leaf epidermis to prevent penetration of UV radiation. [START_REF] Chapple | An Arabidopsis Mutant Defectivein the General Phenylpropanoid Pathway[END_REF][START_REF] Ruegger | Mutations that Reduce Sinapoyl Malate Accumulation in Arabidopsis Thaliana Define Loci with Diverse Roles in Phenylpropanoid Metabolism[END_REF][START_REF] Fraser | The Phenylpropanoid Pathway in Arabidopsis[END_REF] Sinapoyl malate is an ester derivative of sinapic acid. Both of them can efficiently dissipate the energy into their surroundings. [START_REF] Baker | Ultrafast Photoprotecting Sunscreens in Natural Plants[END_REF] As a result, an interesting question is why nature selects sinapoyl malate rather than sinapic acid as the plant sunscreen? Sinapoyl malate in the gas phase exhibits a more broadening absorption spectrum in the UVB region than sinapic acid, which is suggested to be the reason for nature's selection of sinapoyl 4 malate as the sunscreen molecule. [START_REF] Dean | Plant Sunscreens in the UV-B: Ultraviolet Spectroscopy of Jet-Cooled Sinapoyl Malate, Sinapic Acid, and Sinapate Ester Derivatives[END_REF] However, in dioxane, acetonitrile and methanol, the two molecules both exhibit high extinction coefficients in the UVB region and thus the reason for the selection is still under discussion. [START_REF] Baker | Ultrafast Photoprotecting Sunscreens in Natural Plants[END_REF] In addition, the excited-state relaxation mechanisms of sinapic acid and sinapoyl malate in the gas phase differ from that in organic solvents. In the gas phase, the initially populated ππ* states are suggested to relax to long-lived nπ* states through internal conversion. [START_REF] Dean | Plant Sunscreens in the UV-B: Ultraviolet Spectroscopy of Jet-Cooled Sinapoyl Malate, Sinapic Acid, and Sinapate Ester Derivatives[END_REF] However, a femtosecond study suggests that in organic solvents the ππ* states relax to the ground state (S 0 ) via an ultrafast trans-cis photoisomerization. [START_REF] Baker | Ultrafast Photoprotecting Sunscreens in Natural Plants[END_REF] The trans-cis photoisomerization is also found in the relaxations of the closely related para-methoxy methylcinnamate (p-MMC) in aqueous solution, [START_REF] Tan | Excited-State Dynamics of Isolated and Microsolvated Cinnamate-Based UV-B Sunscreens[END_REF][START_REF] Miyazaki | Direct Spectroscopic Evidence of Photoisomerization in para-Methoxy Methylcinnamate Revealed by Low-Temperature Matrix-Isolation FTIR Spectroscopy[END_REF] as well as sinapic acid in aqueous solution. [START_REF] Vengris | Ultrafast Dynamics of Isolated Model Photoactive Yellow Protein Chromophores: "Chemical Perturbation Theory" in the Laboratory[END_REF] However, the relaxation mechanism of sinapoyl malate in aqueous solution is unknown.

Herein, we explore the excited-state dynamics of sinapic acid and sinapoyl malate in aqueous solution. In pH = 6~8 aqueous solution, the carboxyl group is deprotonated and the phenolic hydroxyl group is protonated. [START_REF] Smyk | Spectroscopic Investigation of Thee Quilibria of the Ionic Forms of Sinapic Acid[END_REF] Thus, sinapic acid and sinapoyl malate exist as SA -and SM 2- species, respectively, as shown in Figure 1. The protonated SA species is also studied under acidic environment in order to provide a guideline for the chemical modification of sinapic acid, such as methyl sinapate. [START_REF] Baker | Ultrafast Photoprotecting Sunscreens in Natural Plants[END_REF][START_REF] Dean | Plant Sunscreens in the UV-B: Ultraviolet Spectroscopy of Jet-Cooled Sinapoyl Malate, Sinapic Acid, and Sinapate Ester Derivatives[END_REF] The potentials of SA, SA -and SM 2-as sunscreen molecules are investigated using static absorption and femtosecond transient absorption (FTA) spectroscopy, as well as time-dependent density functional theory (TD-DFT). Note that the abbreviation SA only represents the protonated form of sinapic acid in acidic environment. The ultrafast barrierless photoisomerization of the three species are identified. The electronic absorption properties of their cis-photoproducts are also discussed.

Sinapic acid (≥ 98%, Sigma-Aldrich) was dissolved in water and hydrochloric acid (~ 1mM) to obtain the SA -and SA aqueous solution, respectively. Sinapoyl malate was synthesized as 5 described in previous studies. [START_REF] Allais | Straightforward Total Synthesis of 2-O-Feruloyl-Lmalate, 2-O-Sinapoyl-L-malate and 2-O-5-Hydroxyferuloyl-L-malate[END_REF] The high-purity powder was dissolved in water and thus the SM 2- aqueous solution was obtained. The three species were confirmed by UV-Vis absorption spectroscopy measurements (Perkin-Elmer). The FTA setup was used as described in previous studies. [START_REF] Yang | Temperature Effect on the Excited-state Dynamics 2-Methyl-5-t-butylp-quaterphenyl: Three-emission-state mode[END_REF][START_REF] Yang | Effects of Solvent Dielectric Constant and Viscosity on Two Rotational Relaxation Paths of Excited 9-(Dicyanovinyl) Julolidine[END_REF] Briefly, the pump pulse was 320 nm for SA, 305 nm for SA -, and 325 nm for SM 2-.

The powers of these excitation pulses were about 0.5 mW. Probe pulse was drawn from a broadband white-light continuum (400-700 nm), generated by focusing a weak 800 nm pulse into a sapphire. The polarization of the pump pulse was set to the magic angle (54.7 o ) relative to the probe pulse. To avoid the concentration of cis-products induced by UV excitation, transient absorption spectra were taken using a flow cell. The UV-Visible spectra before and after FTA measurements were shown in Figure S1 (see the supporting information, SI). Continuous UV irradiation studies were performed on the three solutions using a nanosecond 355 nm pulse with the power of 3 mW in a 10mm * 10mm quartz cuvette. The 355 nm pulse was chosen because it was near the center of UVA light. The UV-Vis absorption spectra were taken to probe the changes upon photoexcitation.

All the ab initio calculations were performed using the Gaussian09 program. [START_REF] Frisch | Gaussian 09[END_REF] Default parameters were used unless otherwise indicated. TD-DFT with the M062X functional [START_REF] Zhao | Density Functionals with Broad Applicability in Chemistry[END_REF] was employed, combined with the linear-response polarized continuum model (LR-PCM) [START_REF] Tomasi | Quantum Mechanical Continuum Solvation Models[END_REF][START_REF] Cossi | Time-Dependent Density Functional Theory for Molecules in Liquid Solutions[END_REF] The steady-state UV-Vis absorption and fluorescence spectra of SA, SA -and SM 2-are shown in Figure 2. All the three species have large absorption section at UVB and high-energy UVA regions. The absorption spectrum of SA -is peaked at 304 nm and its intensity decreases to zero at approximately 360 nm. Comparatively, the absorption spectra of SA and SM 2-are slightly red shifted. Their absorption maxima are both at 320 nm and they absorb UVA light till approximately 380 nm. The more broadening absorption of SA and SM 2-relative to SA -makes them behave as better sunscreen molecules. The fluorescence spectra of SA and SM 2-are both peaked at approximately 450 nm, also slightly red shifted compared to the fluorescence spectrum of SA -, which is peaked at approximately 425 nm. The deprotonation state of sinapic acid affects its electronic absorption characteristics. [START_REF] Vengris | Ultrafast Dynamics of Isolated Model Photoactive Yellow Protein Chromophores: "Chemical Perturbation Theory" in the Laboratory[END_REF] For example, deprotonating the carboxylic group of sinapic acid results in the absorption maximum blue shifted slightly from 320 to 304 nm. In contrast, further deprotonating the phenolic hydroxyl group (SA 2-) makes the absorption maximum red shifted largely to 350 nm, as shown in Figure S2. This implies that deprotonating the carboxylic group and phenolic hydroxyl group have exactly the opposite effects on the 7 electronic excitation energy of sinapic acid. This phenomenon is also observed in sinapoyl malate. Deprotonating the carboxyl group of sinapoyl malate only leads to the absorption maximum blue shifted slightly from 328 to 320 nm, while further deprotonating the phenolic hydroxyl group (SM 3-) leads to the absorption maximum largely red shifted to 380 nm (Figure S2). The small absorption cross section in UVB region makes SA 2-and SM [START_REF] Cadet | DNA Base Damage by Reactive Oxygen Species, Oxidizing Agents, and UV Radiation[END_REF]-not suitable to serve as sunscreen molecules. Figure 3 shows the FTA spectra of SA, SA -and SM 2-for photoexcitation at 320, 305 and 325 nm, respectively. For SA, the FTA spectra are dominated by a broad feature spanning the spectral region of ~ 400-500 nm centered at 450 nm. This is attributed to the stimulated emission, which is in line with the steady-state fluorescence (Figure 2). [START_REF] Baker | Ultrafast Photoprotecting Sunscreens in Natural Plants[END_REF][START_REF] Dean | Plant Sunscreens in the UV-B: Ultraviolet Spectroscopy of Jet-Cooled Sinapoyl Malate, Sinapic Acid, and Sinapate Ester Derivatives[END_REF][START_REF] Vengris | Ultrafast Dynamics of Isolated Model Photoactive Yellow Protein Chromophores: "Chemical Perturbation Theory" in the Laboratory[END_REF] In this region, the absorption of 8 solvated electron and phenoxyl-type radical species can be ignored due to the strong absorption intensity of solutes (Figure S1) and much shorter lifetime relative to the radicals. [START_REF] Vengris | Ultrafast Dynamics of Isolated Model Photoactive Yellow Protein Chromophores: "Chemical Perturbation Theory" in the Laboratory[END_REF] At 0.2 ps, the stimulated emission is peaked at ~ 430 nm and then it red shifts to ~ 450 nm at 1 ps, induced by the intramolecular vibrational energy redistribution (IVR) and solvation relaxation. The negative absorption finally decays to the baseline by ~ 20 ps. The FTA spectra for SA -and SM 2-are also dominated by their negative stimulated emissions. Obviously, SA -decays slower than the other two molecules. To give a quantitative insight into their dynamical relaxations, the decays of their stimulated emissions are fitted by mono-or bi-exponential functions. The lifetimes of the three species are summarized in Table 1. A bi-exponential function fits well the decays of SA and SA -.

In SA, the τ 1 (0.28 ps) with a positive pre-exponential factor is assigned to the coupled IVR and solvation relaxation processes, while the τ 2 (6.1 ps) with a negative pre-exponential factor is assigned to the decay of ππ*state. The ππ*state might relax to a dark nπ* state like in the gas phase and/or decay back to the S 0 state via trans-cis photoisomerization like cinnamate derivatives, [START_REF] Miyazaki | Direct Spectroscopic Evidence of Photoisomerization in para-Methoxy Methylcinnamate Revealed by Low-Temperature Matrix-Isolation FTIR Spectroscopy[END_REF][START_REF] Dean | Plant Sunscreens in the UV-B: Ultraviolet Spectroscopy of Jet-Cooled Sinapoyl Malate, Sinapic Acid, and Sinapate Ester Derivatives[END_REF] which will be discussed further by ab initio calculations. In addition, the assignment strategy is also reasonable for the fitted lifetimes of SA -. The 23.5 ps lifetime of SA - is in good agreement with the 26 ps lifetime measured by Vengris et al. [START_REF] Vengris | Ultrafast Dynamics of Isolated Model Photoactive Yellow Protein Chromophores: "Chemical Perturbation Theory" in the Laboratory[END_REF] The τ 1 of SA -(1.1 ps) is much longer than that of SA (0.28 ps), indicating that the IVR and solvation relaxation processes are much slower for SA -. This can be also seen from their wavelength evolutions (Figures 3a,3b). The decay of SM 2-is fitted by a mono-exponential function and τ 1 = 16.9 ps is obtained, which corresponds to the decay of ππ*state. The IVR and solvation relaxation processes are absent in the fitting results. Based on the similar wavelength evolutions of SA and SM 2-(Figures 3a,3c), it can be inferred that the lifetime of IVR and solvation relaxation process is at sub-ps timescale. Although the ππ* state relaxation of SA -is the slowest, it cannot conclude Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083 9 that SA -is not a suitable sunscreen molecule, because the lifetimes of the other two molecules are also at the same timescale. There should be other reasons for nature's selection of SM 2-as the sunscreen molecule. 10 these equilibrium geometries, the vertical excitation energies and oscillator strengths of the S 1 and S 2 were calculated at the LR-PCM/TD-M062X/6-31+g(d, p) level, as collected in Table 2.

For all the trans-species, the S 1 state is bright ππ* state and the S 2 state is dark nπ* state. After considering the intermolecular hydrogen bonding interactions with water, the results do not have significant changes, as shown in Figure S4 and Table S1. This energy level sequence is contrast with the case in the gas phase due to the strong polar aqueous environment stabilizing the ππ* state and destabilizing the nπ* state. [START_REF] Tan | Excited-State Dynamics of Isolated and Microsolvated Cinnamate-Based UV-B Sunscreens[END_REF][START_REF] Dean | Plant Sunscreens in the UV-B: Ultraviolet Spectroscopy of Jet-Cooled Sinapoyl Malate, Sinapic Acid, and Sinapate Ester Derivatives[END_REF] The calculated excitation energies are overestimated compared to the experimental absorption maxima. However, the calculated results are in qualitative agreement with experiment. The electronic absorption energy of trans-SA -is the highest while that of trans-SA and trans-SM 2-are similar.

In order to evaluate the possibility of the ππ*-nπ* internal conversion, the nπ* geometries of trans-SA, SA -and SM 2-are optimized and their vertical electronic transitions are calculated, as shown in Table S2. For trans-SA and SM 2-, the nπ* state is still described as the S 2 state while the S 1 state is the ππ* state, indicating that the nπ* state always lies in a higher energy level than the ππ* state. Thus, the ππ*-nπ* internal conversion cannot occur. In contrast, the ππ*-nπ* internal conversion of trans-SA -is possible because the nπ* state is more stable than the ππ* state in the equilibrium nπ* geometry of trans-SA -. However, FTA spectra suggest that the excited-state evolutions of SA -and SA are very similar. In addition, in less polar organic solvents where the nπ* state is more stable than in aqueous solution, [START_REF] Liu | Hydrogen Bond Dynamics Governs the Effective Photoprotection Mechanism of Plant Phenolic Sunscreens[END_REF] there is no significant evidence to support the ππ*-nπ* internal conversion. [START_REF] Baker | Ultrafast Photoprotecting Sunscreens in Natural Plants[END_REF] As a result, the role of the nπ* state in the ππ* state relaxations can be ignored. This is in a good agreement with the results that the hydration of p-MMC impedes the ππ*-nπ* internal conversion. 24 11 Now that the ππ*-nπ* internal conversion is unlikely to occur, in order to confirm the ππ* relaxation through the trans-cis photoisomerization, the potential energy surfaces (PESs) of the photoisomerization of SA, SA -and SM 2-are constructed by LIIC method at the LR-PCM/TD-M062X/6-31+g(d, p) calculation level, as shown in Figure 4. The isomerization paths of the three species are all barrierless, indicating that the photoisomerization occurs dynamically on PES, in agreement with the photoisomerization of the closely related p-MMC. [START_REF] Miyazaki | Direct Spectroscopic Evidence of Photoisomerization in para-Methoxy Methylcinnamate Revealed by Low-Temperature Matrix-Isolation FTIR Spectroscopy[END_REF] Note that for every point in the three PESs, the S 1 state is always the ππ* state. The barrierless relaxation path is in good agreement with the ultrafast decays of ππ* states, which are completed less than tens of picoseconds. Although TD-DFT is unable to locate the conical intersection (CI), it can give a qualitative description if the energy gap between the S 0 and S 1 states is small enough. [START_REF] Gustavsson | Singlet Excited-state Behavior of Uracil and Thymine in Aqueous Solution: A Combined Experimental and Computational Study of 11 uracil derivatives[END_REF] Thus, the PESs shown in Figure 4 are believable. After initial photoexcitation, the ππ* state evolves along the barrierless photoisomerization path characterized by the decreasing η. When the ππ* state reaches the low-lying ππ*-S 0 CI, η is 80 o for SA -and 90 o for both SA and SM 2-. At this point, ultrafast nonradiative decay to the S 0 state is extremely efficient. The system can either evolve to the cis-product or reverse back to the original trans-reactant. In addition, the reverse cis-trans isomerization is also barrierless, indicating that there is a dynamical equilibrium between the trans-cis and cis-trans reactions, similar to the case of p-MMC. As has been discussed, cis-photoproducts can be formed by nonradiative trans-cis photoisomerziation. [START_REF] Baker | Ultrafast Photoprotecting Sunscreens in Natural Plants[END_REF] To evaluate the effect of the formation of cis-products on the efficacy of sunscreen molecules, a continuous UV irradiation study is performed using the 355 nm pulse with a power of 3 mW. Steady-state UV-Vis spectroscopy is used to probe the absorption changes induced by the formation of cis-photoproducts. As shown in Figure 5b, after 355 nm 13 light irradiation, the absorption spectra of SA -are blue shifted significantly accompanied with largely decreasing intensities. This will largely reduce the efficacy of SA -as a sunscreen molecule. The spectral changes of SA -are in good agreement with the TD-DFT results shown in Table 2. TD-DFT results suggest that electronic excitation energy of cis-SA -is higher than that of trans-SA -and the oscillator strength of the former is smaller. As to SA and SM 2-, the absorption spectra after the 355 nm light irradiation show no spectral shift and their intensities only decrease slightly, in agreement with the TD-DFT results that trans-SA and cis-SA have the same electronic excitation energies (Table 2). Although cis-SM 2-has 0.13 eV higher excitation energy than trans-SM 2-, the TD-DFT results is generally in good agreement with the irradiation results. As a result, SA and SM 2-are better sunscreen molecules than SA -.

In conclusion, we have studied the photoprotection mechanism of SA, SA -and SM 2-in aqueous solution. The initially populated ππ* states of the three species all decay to the S 0 state nonradiatively via a barrierless trans-cis photoisomerization, which are completed in a few tens of picoseconds. The photoisomerization leads to the formation of cis-photoproducts. Cis-SA -has much higher excitation energy and a decreased absorption cross section in the UV light than trans-SA -. Thus, the efficacy of SA -as sunscreen is reduced largely after photoexcitation. In contrast, the absorption spectra of SA and SM 2-after photoisomerization just show slightly decreased intensities. These results suggest that SM 2-is a better sunscreen molecule than SA -, explaining nature's selection of sinapoyl malate rather than sinapic acid as the plant sunscreen.

In addition, the protonated SA exhibits an ultrafast excited-state lifetime and its cis product still has a large absorption cross section on the UV region. An appropriate chemical modification on SA can make it a good sunscreen molecule used in neutral pH condition. These results highlight

KEYWORDS

  Femtosecond, TDDFT, Sinapic Acid, Photochemistry, Excited State. Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083

Figure 1 .

 1 Figure 1. Chemical structures of sinapic acid in acidic environment (SA) and neutral environment (SA -), as well as the chemical structures of sinapoyl malate in acidic environment (SM) and neutral environment (SM 2-).

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 17 Paragon Plus Environment The Journal of Physical Chemistry Letters Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083

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 17 Paragon Plus Environment The Journal of Physical Chemistry Letters Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083

6 Figure 2 .

 62 Figure 2.Normalized steady-state absorption (left) and fluorescence (right) spectra of SA, SA - and SM 2-in aqueous solution.

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 17 Paragon Plus Environment The Journal of Physical Chemistry Letters Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083

Figure 3 .

 3 Figure 3. Femtosecond wavelength evolution curves of (a) SA, (b) SA -, and (c) SM 2-. Fitting results of the decay of (d) SA at 430 nm, (e) SA -at 430 nm, and (f) SM 2-at 453 nm. Black

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 17 Paragon Plus Environment The Journal of Physical Chemistry Letters Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083

S 0

 0 state via trans-cis photoisomerization, TD-DFT calculations are carried out. The S 0 geometries of trans-SA, SA -and SM 2-are optimized, as shown in Figure S3. The dihedral angle η (C 1 -C 2 -C 3 -C 4 ), defined to describe the double bond rotation, is 180 o for all the three species. Based on Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083

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 17 Paragon Plus Environment The Journal of Physical Chemistry Letters Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083

6 Page 11 of 17 ACS 12 Figure 4 .

 617124 Figure 4. LR-PCM/TD-M062X/6-31+g(d, p) calculated linearly interpolated internal coordinate (LIIC) paths of the trans-cis photoisomerization of (a) SA, (b) SA -, and (c) SM 2-. Dihedral angle η (C 1 -C 2 -C 3 -C 4 ) is also represented. In the original trans-reactant, the dihedral angle is 180 o . And in the final cis-product, the dihedral angle is near to 0 o .

Figure 5 .

 5 Figure 5. The absorption spectra of (a) SA, (b) SA -, and (c) SM 2-after a period of 355 nm pulse

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 17 Paragon Plus Environment The Journal of Physical Chemistry Letters Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083

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 17 Paragon Plus Environment The Journal of Physical Chemistry Letters Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083 14 the role of the photoproduct in the efficacy of a sunscreen molecule, providing a new criterion for designing a new sunscreen molecule. ASSOCIATED CONTENT Supporting Information. Computational details, steady-state UV-Vis absorption spectra of sinapic acid and sinapoyl malate in NaOH solution, optimized ground-state geometries, vertical excitation energies of equilibrium nπ* geometries. This material is available free of charge via the Internet at http://pubs.acs.org. Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083 Comment citer ce document : Luo, J., Liu, Y., Yang, S., Flourat, A., Allais, F., Han, K. (Auteur de correspondance) (2017). Ultrafast barrierless photoisomerization and strong ultraviolet absorption of photoproducts in plant sunscreens. Journal of Physical Chemistry Letters, 8 (5), 1025-1030. DOI : 10.1021/acs.jpclett.7b00083 TOC 76x31mm (600 x 600 DPI)

Table 1 .

 1 Summary of the excited-state lifetimes of SA, SA -and SM 2-based on their decays of

	stimulated emissions
		SA	SA -	SM 2-
	a 1	0.46±0.08	0.27±0.05	-46.3±1.0
	τ 1 /ps	0.28±0.09	1.1±0.4	16.9±1.1
	a 2	-1.14±0.03	-0.49±0.04
	τ 2 /ps	6.1±0.4	23.5±3.0

Table 2 .

 2 Vertical

		E (SA)/eV	E (SA -)/eV	E (SM 2-)/eV
	trans-S 1	4.05 (0.69)	4.46 (0.64)	4.12 (0.73)
	trans-S 2	4.51 (0.03)	4.73 (0.00)	4.62 (0.02)
	cis-S 1	4.03 (0.57)	4.78 (0.46)	4.25 (0.39)
	In order to determine whether the initially populated ππ* state decays to dark nπ* state or the

excitation energies (in eV) including the first and second excited states (S 1 , S 2 ) of trans-SA, SA -and SM 2-at the LR-PCM/TD-M062X/6-31+g(d, p) level, as well as the S 1 state of their respective cis-forms. Oscillator strengths are shown in the parentheses.

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Paragon Plus Environment The Journal of Physical Chemistry Letters ACKNOWLEDGMENT We are grateful to the National Basic Research Program of China (2013CB834604) and the National Natural Science Foundation of China (Grant No: 21533010).

AUTHOR INFORMATION

Corresponding Author

*Email: klhan@dicp.ac.cn

Notes

#

These authors contributed equally to this work.

The authors declare no competing financial interests.

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The Journal of Physical Chemistry Letters

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The Journal of Physical Chemistry Letters