Selenium supplementation via modulation of selenoproteins ameliorates binge drinking- induced oxidative, energetic, metabolic, and endocrine imbalance in adolescent rats’ skeletal muscle

Food & Function PAPER Cite this: Food Funct., 2024, 15, 7988 Received 22nd March 2024, Accepted 26th June 2024 DOI: 10.1039/d4fo01354a rsc.li/food-function Selenium supplementation via modulation of selenoproteins ameliorates binge drinkinginduced oxidative, energetic, metabolic, and endocrine imbalance in adolescent rats’skeletal muscle Inés Romero-Herrera, a Fátima Nogales, * a María del Carmen Gallego-López, a Javier Díaz-Castro, b,c Olimpia Carreras a and María Luisa Ojeda a Adolescence is characterized by increased vulnerability to addiction and ethanol (EtOH) toxicity, particularly through binge drinking (BD), a favored acute EtOH-ingestion pattern among teenagers. BD, highly pro-oxidant, induces oxidative stress (OS), affecting skeletal muscle (SKM), where selenium (Se), an antioxidant element and catalytic center of selenoproteins, is stored, among other tissues. Investigating the effects of Se supplementation on SKM after BD exposure holds therapeutic promise. For this, we randomised 32 adolescent Wistar rats into 4 groups, exposed or not to intermittent i.p. BD [BD and control (C)] (3 g EtOH per kg per day), and supplemented with selenite [BDSe and CSe] (0.4 ppm). In SKM, we examined the oxidative balance, energy status (AMPK, SIRT-1), protein turnover (IRS-1, Akt1, mTOR, IGF-1, NFκB p65, MAFbx, ULK1, pelF2α), serum myokines (myostatin, IL-6, FGF21, irisin, BDNF, IL-15, fractalkine, FSTL-1, FABP-3), and selenoproteins (GPx1, GPx4, SelM, SelP). In the pancreas, we studied the oxidative balance and SIRT-1 expression. Selenite supplementation mitigated BD-induced OS by enhancing the expression of selenoproteins, which restored oxidative balance, notably stimulating protein synthesis and normalizing the myokine profile, leading to improved SKM mass growth and metabolism, and reduced inflammation and apoptosis (caspase-3). Selenite restoration of SelP’s receptor LRP1 expression, reduced by BD, outlines the crucial role of SKM in the SelP cycle, linking Se levels to SKM development. Furthermore, Se attenuated pancreatic OS, preserving insulin secretion. Se supplementation shows potential for alleviating SKM damage from BD, with additional beneficial endocrine effects on the pancreas, adipose tissue, liver, heart and brain that position it as a broad-spectrum treatment for adolescent alcohol consumption, preventing metabolic diseases in adulthood. 1. Introduction Adolescence is a period marked by intense developmental, endocrine, and metabolic changes, characterized by impulsive behavior attributed to the immaturity of the prefrontal cortex. 1 Therefore, addictive disorders related to substances and/or behaviors typically begin during adolescence or early adulthood. 2 Besides, this age group is especially susceptible to the toxicity of ethanol (EtOH); 3–8 in this context, binge drinking (BD) is a pattern of acute EtOH consumption that has lately been prominently favored by teenagers, 9,10 consisting of reaching a blood alcohol concentration of 0.08% or higher within 2 hours. 11 The harmfulness of BD resides in its pronounced pro-oxidant effect on DNA, proteins and lipids, as it greatly induces the hepatic activity of cytochrome P450 2E1 (CYP2E1), much more than chronic alcoholism. 12 CYP2E1 generates a large amount of reactive oxygen species (ROS) to metabolize the excess of EtOH ingested, leading to oxidative stress (OS) when the antioxidant defense capacity of the cell is surpassed. 13,14 NADPH oxidase 4 (NOX4) is another wellknown EtOH-induced source of ROS and OS, whose activity increases serum hydrogen peroxide levels (H 2 O 2 ). 6,15 a Department of Physiology, Faculty of Pharmacy, University of Seville, C/Professor García González 2, 41012-Seville, Spain. E-mail: iromer[email protected], [email protected], [email protected], [email protected], ojedamuri[email protected] b Institute of Nutrition and Food Technology “José Mataix Verdú”, University of Granada, Avenida del Conocimiento s/n, 18071-Armilla, Granada, Spain. E-mail: [email protected] c Department of Physiology, University of Granada, Granada, Spain 7988 |Food Funct.,2024,15,7988–8007 This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 10 July 2024. Downloaded on 2/26/2025 7:44:28 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue The adverse impact of BD on the central nervous system (CNS) has been pervasively studied; 16,17 however, its detrimental effects on other systems, such as the hepatic, renal or cardiovascular systems, have also been demonstrated, promoting the emergence of cardiometabolic diseases over time. 3,5,18,19 Moreover, in the three principal target organs for insulin action, the liver, skeletal muscle (SKM) and adipose tissue, adolescent rats after BD exposure exhibited dysregulation of two crucial cellular energy sensors, AMP activated protein kinase (AMPK) and NAD + -dependent deacetylase sirtuin 1 (SIRT-1); this can be related to depletion in the expression of the insulin receptor substrate 1 (IRS-1), which was also found. 5,7,8 This situation predisposes adolescents to the development of insulin resistance (IR), 20,21 which is a factor in metabolic syndrome, that has lately become a highly prevalent disease in adolescents. 22,23 SKM is a tissue that stands out in this context for being the primary site for glucose disposal in response to insulin or exercise; 24 it can also store glucose and serves as the reservoir of major metabolic amino acid, to be used in case of energy requirements. Regrettably, BD during adolescence has recently been linked to impaired SKM protein turnover. 7,25 Furthermore, OS in SKM leads to AMPKαstimulation, 7,26 which induces a catabolic state that inhibits protein synthesis routes, such as the IRS-1/PI3K/Akt pathway of the mammalian target of rapamycin complex 1 (mTORC1); and activates the degradation mechanisms, the ubiquitin-proteasome (UPS) and autophagic-lysosomal systems, via stimulation of atrogin-1 (or muscle atrophy F-box, MAFbx) and Unc-51-like autophagy activating kinase 1 (ULK1), respectively. 7,25 Additionally, although predominantly secreted by the liver, insulin-like growth factor 1 (IGF-1) can also be produced locally in SKM, where it governs protein synthesis via stimulation of the mTOR pathway. 27 In the last decade, SKM has been identified as a secretory organ. Thus, cytokines and other peptides produced, expressed, or released by muscle fibers and exerting paracrine, autocrine, or endocrine effects are classified as myokines. It is crucial that SKM has been established with this role, as it provides us with a conceptual basis for understanding how it communicates with other organs, such as adipose tissue, the liver, pancreas, or the brain, to name but a few. 28,29 Furthermore, disbalance of myokines has been related not only to SKM pathology, but also to general metabolic diseases and to OS. 7,30,31 Given that it has been reported that OS partially mediates the impairment caused by BD, 7 an antioxidant treatment appears to be a suitable therapeutic approach. Selenium (Se) is an essential trace element with renowned antioxidant properties that is the catalytic center of selenoproteins, such as the glutathione peroxidase family (GPx) or selenoprotein P (SelP). 32 Our research team has discovered through several studies that during adolescence, BD alters Se homeostasis and Se tissue distribution, 19 and that dietary Se supplementation is an effective strategy to mitigate BD-induced liver, 5,33,34 kidney 3 and cardiovascular damage; 18 attributed to the ability of Se to reduce oxidative imbalance by upregulating the expressions of GPx1 and GPx4, and by modulating nuclear factor kappa B (NF-κB) activation and apoptosis-related caspase-3 protein in these organs. Selenoproteins differ with regard to their location and function within cells. GPx1 and SelP are cytosolic; GPx1 is involved in redox regulation, reducing H 2 O 2 to water, and is also related to IR; 33 SelP also has antioxidant activity, being mainly responsible for the plasmatic transport of Se from the liver to target organs, thus indirectly controlling the tissue expression of all selenoproteins. This transport is mediated through receptors in each organ: e.g., LRP1 (lowdensity lipoprotein receptor-related protein 1) mediates the uptake of SelP in SKM. 35 GPx4 has an essential role in mitochondria, preventing their oxidation, NF-κB inflammatory response and apoptosis; this selenoprotein eliminates intracellular lipid hydroperoxides, also being implicated in ferroptosis, a regulated cell death characterized by iron-dependent lipid peroxidation. Finally, we find endoplasmic reticulum (ER)-resident selenoproteins, such as selenoprotein M (SelM), which controls ER homeostasis, protecting it through scavenging excessive ROS and suppressing apoptosis. 36 In the ER, excessive ROS produce ER stress, leading to the accumulation of unfolded proteins and thus activating the unfolded protein response (UPR). 37 SKM harbors approximately half of the body’s total Se content, 38 serving as a reservoir for the body when there are deficiencies, such as those produced by BD-induced OS. Consequently, it could be crucial to explore Se dietary supplementation in SKM after BD exposure. Therefore, the aim of this study is to analyze the oxidative, metabolic, energetic, and endocrine balance after BD exposure in the SKM of adolescent rats receiving Se supplementation, exploring a possible mechanism of action through selenoprotein modulation and synergic activity. 2. Materials and methods 2.1. Animals Animal care procedures and experimental protocols were performed in accordance with European Union regulations (Directive 2010/63/UE) and with a Spanish Royal Decree (BOE 34/11 370, 2013), which establishes the basic rules applicable for the protection of animals used in experimentation and other scientific purposes. The experiments were also reviewed and approved by the Ethics Committee of the University of Seville (CEEA-US2019-4) and the Andalusian Regional Government (05-04-2019-065). They were meticulously planned and executed with the goal of minimizing unnecessary pain and suffering, adhering to the principles of replacement, reduction, and refinement wherever possible to decrease both the total number of animals and their use. In this experiment, 32 adolescent male Wistar rats (Centre of Production and Animal experimentation, Vice-rector’sOffice for Scientific Research, University of Seville) were used. Rats were received at 21 days of age and housed in groups of 2 rats Food & Function Paper This journal is © The Royal Society of Chemistry 2024 Food Funct.,2024,15,7988–8007 | 7989 Open Access Article. Published on 10 July 2024. Downloaded on 2/26/2025 7:44:28 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online per cage for 1 week, to acclimatize them to the housing conditions and handling. The experimental treatment took place during a 3-week period, starting at postnatal day (PND) 28 and ending at PND 46 (sacrifice). This period corresponds to adolescence in Wistar rats. 39 The animals were kept at an automatically controlled temperature (22–23 °C) and in a 12 : 12 h light/dark photocycle (light: 07:00 a.m. to 19:00 p.m.). On PND 28, when the adolescent period began, rats were randomly assigned into 4 groups (n= 8 per group) according to their treatment: control group (C), which received a standard pellet diet and drinking water ad libitum, and on the corresponding days, an isotonic physiological saline solution (PSS) intraperitoneally (i.p.); BD alcohol group (BD), which received a standard pellet diet and drinking water ad libitum, and on the corresponding days, an ethanol solution in PSS i. p.; control Se group (CSe), which received a standard pellet diet and Se supplementation in drinking water ad libitum, and on the corresponding days an injection of PSS i.p.; and BD alcohol Se group (BDSe), which received a standard pellet diet and drinking water supplemented with Se ad libitum, and on the corresponding days, an alcohol solution in PSS i.p. The standard pellet diet (2014 Teklad Global 14% Protein Rodent Maintenance Diet, Harlan Laboratories, Barcelona, Spain) contained 0.23 ppm of Se. The Se-supplemented groups (CSe and BDSe) received 0.14 ppm of extra Se as anhydrous sodium selenite (PanReac, Barcelona, Spain) in drinking water over the whole experimental period. 2.2. Nutritional control Body weight and the amount of food consumed by the rats were monitored daily until the end of the experimental period. The amount of food ingested every day was calculated by measuring this parameter every morning and the next day; the difference between them was the amount consumed. Total Se intake was calculated by multiplying the known Se concentration in the diet (0.23 ppm) and in the drinking water (0.14 ppm for CSe and BDSe groups) by the amounts of food and water ingested every day. All measurements were taken at 9:00 to avoid changes due to circadian rhythms. 2.3. Ethanol treatment Alcohol BD-exposed groups (BD and BDSe) received an i.p. injection of alcohol in PSS (20% v/v) with a dose of 3 g/ kg −1 day −1 . Alcohol injections were given starting at 19:00, when the dark cycle began, for 3 consecutive days each week for 3 weeks (a total of 9 injections for each rat). No i.p. injections were given during the remaining 4 days of each week. This BD model is one of the most commonly used since it easily ensures a blood alcohol concentration of 80 mg dL −1 , the value established by the NIAAA for BD. 11 In addition, this method of alcoholization in adolescent rats has previously been used by this research group, registering a blood alcohol concentration of almost 125.0 mg dL −1 1 h after the last injection. 12 The control groups (C and CSe) received an i.p. injection of an equal volume of PSS at the same time as the injections to the alcohol BD-exposed group. 2.4. Samples At the end of the experimental period (PND 46), the rats were fasted for 12 h (from 07:00 to 19:00). Then, 24 h after their last EtOH exposure or treatment with saline solution (last injection: PND 45, 19:00), the adolescent rats were anesthetized with an i.p. injection of 28% w/v urethane (0.5 ml per 100 g of body weight) and sacrificed (PND 46, 19:00). The blood was obtained by heart puncture and collected in tubes. The serum was prepared using low-speed centrifugation for 15 min at 1300g. The abdomen was opened by a midline incision to extract the pancreas; SKM gastrocnemius was obtained from the right leg. Once removed, both organs were immediately frozen in liquid nitrogen and stored at −80 °C. 2.5. Biochemical measurements in serum In serum, the creatinine, creatine phosphokinase (CPK) and insulin were measured with an automated analyzer (Technicon RA-1000, Bayer Diagnostics, Leverkusen, Germany). 2.6. Antioxidant enzyme activity and oxidative stress markers In order to measure the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as the lipid and protein oxidation, pancreas and SKM tissue samples were homogenized (25 000 rpm for 1 min, 1 : 4 w/v) using the homogenizer Fisherbrand™ 850 (Thermo Fisher Scientific Inc., Waltham, MA, USA) in a sucrose buffer (15 mM Tris/HCl, pH 7.4, 250 mM sucrose, 1 mM EDTA, and 1 mM dithiothreitol) in an ice bath. The homogenates were centrifuged at 900gfor 20 min at 4 °C, and the final supernatant was aliquoted and frozen at −80 °C until analysis. The activity of SOD (U mg −1 protein) was determined by the Fridovich method (1985), 40 which is based on the ability of SOD to inhibit the reduction of cytochrome c induced by the xanthine–xanthine oxidase system; it is measured by the absorbance increase at 550 nm for 3 min, due to the reduction of cytochrome c by adding xanthine oxidase. CAT activity (U mg −1 protein) was determined using H 2 O 2 as substrate by the assay of Beers and Sizer (1952), 41 where the disappearance of H 2 O 2 was followed spectrophotometrically at 240 nm for 3 min. The GPx activity (mU mg −1 ) was determined using the method described by Lawrence and Burk (1976). 42 In this assay, the oxidized glutathione (GSSG) formed by the action of GPx is coupled to the reaction that catalyzes the glutathione reductase (GR) enzyme, measuring the absorbance decrease at 340 nm for 3 min due to the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH). In order to measure lipid peroxidation in the pancreas and SKM, we quantified the malondialdehyde (MDA) levels (mol mg −1 protein), the end-product of the oxidative degradation of lipids, using a colorimetric reaction with thiobarbituric acid (TBA) at 535 nm, as described by Draper and Hadley (1990). 43 The protein oxidation in SKM was measured by the detection of carbonyl groups (CG) (nmol mg −1 protein) at 366 nm by the method described by Reznick and Packer (1994). 44 where the Paper Food & Function 7990 |Food Funct.,2024,15,7988–8007 This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 10 July 2024. Downloaded on 2/26/2025 7:44:28 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online reaction of 2,4-dinitrophenylhydrazine (DNPH) with CG takes place. Hydrogen peroxide (H 2 O 2 ) concentration was also determined (µM), this molecule being a reactive oxygen metabolic by-product. For this, we used an H 2 O 2 colorimetric detection kit (catalog number KA1017, Abnova, Taipei, Taiwan). The kit has a color reagent that contains a dye, xylenol orange, in an acidic solution with sorbitol and ammonium iron sulfate that reacts to produce a purple color proportional to the concentration of H 2 O 2 in the sample. The optical density was measured at 550 nm, using a plate reader. 2.7. Protein immunoblotting assays To this end, tissue samples were homogenized (25 000 rpm for 1 min, 1 : 10 w/v) in 50 mm phosphate buffer [K 2 HPO 4 50 mm, KH 2 PO 4 50 mm, EDTA 0.01 mm, protease inhibitor 1 : 10 (Complete Protease Inhibitor Cocktail Tablets, Roche, Mannheim, Germany), phosphatase inhibitor 1 : 10 (PhosSTOP Phosphatase Inhibitor Cocktail Tablets, Roche, Mannheim, Germany)] using the homogenizer Fisherbrand™850 (Thermo Fisher Scientific Inc., Waltham, MA, USA). Then, the homogenates were centrifuged at 500gat 4 °C for 10 min, and the final supernatant was aliquoted and frozen at −80 °C until analysis. The protein expression measurements in our adolescent rats were conducted using western blot, a protein immunodetection technique. In the pancreas, we evaluated the expression of GPx1 and SIRT-1; in SKM, we quantified the expression of Akt1, total-AMPKα(tAMPKα), phospho-AMPKα (Thr172) (pAMPKα), atrogin-1/MAFbx, cleaved caspase-3, phospho-eukaryotic initiation factor 2α(Ser51) (pelF2α), GPx1, GPx4, IGF-1, IRS-1, LRP1, mTOR, phospho-mTOR (Ser2448) (pmTOR), NF-κB p65, NOX4, SelM, SelP, SIRT-1 and UKL1. β-Actin was used as load control for the pancreas and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for SKM. The protein content of the samples was analyzed by the method of Lowry et al. (1951) 45 and the samples utilized contained 100 or 60 μg of protein for the pancreas or SKM, respectively. The calculated appropriate volumes for those protein quantities were mixed with an equal volume of a sample buffer (Laemmli 2× Concentrate, Sigma-Aldrich, Burlington, MA, USA), plus 5% 2-mercaptoethanol (BioRad, CA, USA). After that, the samples were heated at 95 °C for 5 min, to denature the proteins. The proteins were then separated with SDS-polyacrylamide gel electrophoresis (PAGE) using acrylamide gels (6%, 7%, 9%, 10% or 12%, depending on the protein) over 1 h at 150 V; then, they were transferred onto a nitrocellulose membrane (BioRad, CA, USA) using a blot system (Transblot; BioRad, CA, USA) for 2 h at 100 V. Nonspecific membrane sites were blocked for 1 h with a blocking buffer, TBS-M: TBS (50 mM TrisHCl, 150 mM NaCl, 0.1% (v/v) Tween 20, pH 7.5) and milk powder 5% (BioRad, CA, USA); thereafter, they were probed overnight at 4 °C with the specific primary antibodies diluted in TBS-M. The antibodies were all mouse monoclonal IgG from Santa Cruz Biotechnology (Santa Cruz, CA, USA), except pAMPKα and pelF2α, rabbit monoclonal and polyclonal antibodies, respectively from Cell Signaling Technology (Beverly, MA, USA). The primary antibodies diluted in TBS-M were: Akt1 (dilution 1 : 750; catalogue number sc-5298); AMPKα1/2 (1 : 2000; sc-74461); pAMPKα(Thr172) (1 : 4000; #2535); atrogin-1/MAFbx (1 : 750; sc-166806); cleaved caspase-3 (1 : 1000; sc-7272); pelF2α(Ser51) (1 : 750; #9721); GPx1/2 (1 : 1000; sc-133160); GPx4 (1 : 1000; sc-166570); IGF-1 (1 : 750; sc-518040); IRS-1 (1 : 500; sc-8038); LRP1 (1 : 750; sc-57353); mTOR (1 : 1000; sc-517464); pmTOR (Ser2448) (1 : 500; sc293133); NF-κB p65 (1 : 1000; sc-8008); NOX4 (1 : 1000; sc518092); SelM (1 : 1000; sc-514952); SelP (1 : 1000; sc-376858); SIRT-1 (1 : 500; sc-74465); and ULK1 (1 : 500; sc-390904). As loading controls, monoclonal mouse anti β-actin (1 : 10000 in TBS-M; IgG1A5441, Sigma-Aldrich, Burlington, MA, USA) and monoclonal mouse anti GAPDH (1 : 1000 in TBS-M; sc-32233, Santa Cruz, CA, USA) were used to detect β-actin or GAPDH for the pancreas or SKM, respectively. After 48 h incubation of the primary antibodies, the membranes were washed 5 times (5 minutes for each wash) with TBST (TBS + 0.1% Tween 20, PanReac, Barcelona, Spain), and then incubated again, this time with the corresponding secondary antibody (Goat Anti-Mouse IgG (H + L) Horseradish Peroxidase Conjugate, catalogue number 170-6516, BioRad, CA, USA; or Goat Anti-Rabbit IgG (H + L) Horseradish Peroxidase Conjugate, 170-6515, BioRad, CA, USA) diluted in TBS-M. After a 1 h probe, the membranes were washed again 5 times with TBST (5 minutes for each wash). Finally, to reveal the results, we added the commercial developer solution Luminol ECL reagent (GE Healthcare and Lumigen, Buckinghamshire, UK) and introduced the membranes into the image analyzer Amersham Imager 600 (GE Healthcare, Buckinghamshire, UK). Then, quantification of the blots was performed by densitometry with the ImageJ program (National Institutes of Health, Bethesda, Maryland, USA). The results were expressed as percent arbitrary relative units, referring to values in control animals which were defined as 100%. 2.8. Determination of myokines Serum levels of the following myokines were measured: interleukin 6 (IL-6), myostatin, interleukin 15 (IL-15), fractalkine (CX3CL1), fibroblast growth factor 21 (FGF21), irisin, brainderived neurotrophic factor (BDNF), follistatin-like protein 1 (FSTL-1) and fatty acid binding protein 3 (FABP-3). For this, we used a MILLIPLEX® Rat Myokine Panel (Millipore Corp., St Charles, MO, USA), based on immunoassays on the surface of fluorescent-coded beads (microspheres), following the manufacturer’s specifications (50 events per bead, 50 μL sample, gate settings of 8000–15000, time out of 60 seconds, melatonin bead set of 34). The plate was read on a LABScan 100 analyzer (Luminex Corp., Austin, TX, USA) with xPONENT software for data acquisition. Average values for each set of duplicate samples or standards were within 15% of the mean. Myokine concentrations in plasma samples were determined by comparing the mean of duplicate samples with the standard curve for each assay. Serum samples were not diluted for this analysis. Food & Function Paper This journal is © The Royal Society of Chemistry 2024 Food Funct.,2024,15,7988–8007 | 7991 Open Access Article. Published on 10 July 2024. Downloaded on 2/26/2025 7:44:28 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online 2.9. Statistical analysis The results were expressed as the mean ± SD (standard deviation) and analyzed using Prism version 8.0.2 (GraphPad Software Inc., San Diego, CA, USA) statistical analysis software. Statistical differences were assessed using the ordinary twoway analysis of variance (two-way ANOVA). When ANOVA resulted in differences, Tukey’s multiple comparisons post hoc test was conducted to analyze the differences between the means of groups using all possible combinations. The sample size per group was 8 (n= 8 per group). Statistical differences were expressed as p-values, with p< 0.05 considered statistically significant. Throughout the text, p-values are illustrated as follows: vs. C: *p< 0.05, **p< 0.01, ***p< 0.001; vs. BDSe: ● p< 0.05, ●● p< 0.01, ●●● p< 0.001. 3. Results Table 1 shows that total solid intake was similar among groups, while liquid intake was significantly increased in BD rats with respect to C (p< 0.01) and BDSe groups (p< 0.05). In this context, the supplemented groups consumed double the amount of Se compared to the non-supplemented groups (p< 0.001). However, BD and BDSe groups had a lower increased body weight at the end of the experiment than the control groups (p< 0.01). Accordingly, the alcohol groups also presented a lower final body weight (p< 0.05 vs. C; p< 0.01 BDSe vs. CSe). BD rats also presented a lower SKM weight and protein content than C (p< 0.001, p< 0.01) or BDSe rats (p< 0.01, p< 0.001); however, BDSe rats had increased protein content vs. CSe (p< 0.01) and slightly decreased SKM weight against the C group (p< 0.05). Serum CPK and creatinine levels were similar between groups. Even though the pancreas relative mass was similar in the 4 groups studied, in this tissue, BD exposure increased the protein content in both the BD and BDSe groups (p< 0.001). Fig. 1 shows that BD rats had significantly higher SOD (p< 0.001) and CAT (p< 0.001) activities, lower GPx activity (p< 0.001) and higher levels of MDA (p< 0.001), CG (p< 0.001), NOX4 expression (p< 0.001) and H 2 O 2 (p< 0.001). Se supplementation to BD rats significantly decreased SOD activity (p < 0.001), and increased CAT (p< 0.01) and GPx (p< 0.001) activities; it also decreased MDA levels (p< 0.001), H 2 O 2 concentration (p< 0.01) and NOX4 expression (p< 0.001) in SKM. BDSe rats had the highest CAT activity among groups, being higher than in C or CSe (p< 0.001) animals; CG levels and NOX4 expression in BDSe rats were also higher than in C or CSe ones (CAT p< 0.001; NOX4 p< 0.001 vs. C and p< 0.01 vs. CSe). In addition, MDA levels were slightly reduced in the BDSe animals (p< 0.05). The effects of selenium supplementation to C rats were more discrete, it only increased GPx activity (p< 0.001) and NOX4 expression (p< 0.01) vs. C animals. Rats exposed to BD had in SKM significantly lower levels of IRS-1 (p< 0.001), Akt1 (p< 0.01), mTOR (p< 0.001), pmTOR (p < 0.001) and IGF-1 (p< 0.001) expressions than control animals (Fig. 2). Se supplementation to BD rats significantly enhanced all these parameters: IRS-1 (p< 0. 0.001), Akt1 (p< 0.001), mTOR (p< 0.001), pmTOR (p< 0.001) and IGF-1 (p< 0.001). BDSe rats had significant higher levels of Akt1 (p< 0.001) and IGF-1 (p< 0.001) than C or CSe rats, being the most elevated group in these proteins. CSe rats presented increased expressions of IRS-1 (p< 0.001), mTOR (p< 0.001) and pmTOR (p< 0.001) vs. C or BDSe groups, having the highest expression amongst the groups. Fig. 3hows that BD-exposed animals presented in SKM significantly lower expression of tAMPKα(p< 0.001), and higher expression of pAMPKα(p< 0.001), pAMPK/tAMPK ratio (p< 0.001), SIRT-1 (p< 0.001), NF-κB p65 (p< 0.001), atrogin-1/ MAFbx (p< 0.01), ULK1 (p< 0.01) and myostatin (p< 0.01) than control animals. Se supplementation to BD rats significantly increased tAMPKαlevels (p< 0.001), and decreased pAMPKα(p< 0.001), pAMPK/tAMPK ratio (p< 0.001) and NFκB p65 (p< 0.01); however, it increased SIRT-1 expression levels even more (p< 0.001). BDSe rats also had higher levels of tAMPKα(p< 0.01), pAMPKα(p< 0.001), pAMPK/tAMPK ratio (p< 0.01), SIRT-1 (p< 0.001), NF-κB p65 (p< 0.001), atrogin-1/MAFbx (p< 0.01) and ULK1 (p< 0.001) than control Table 1 Nutritional and morphologic parameters; skeletal muscle (SKM) and pancreas profile Parameters C BD CSe BDSe Final body weight (g) 159.2 ± 6.0 143.3 ± 17.2* 159.5 ± 11.0 ●● 142.1 ± 10.7* Increased body weight (g day −1 ) 5.83 ± 0.33 4.85 ± 1.12** 5.85 ± 0.86 ●● 4.73 ± 0.57** Solid intake (mg day −1 ) 15.9 ± 2.0 14.3 ± 1.6 16.3 ± 1.9 14.5 ± 2.5 Liquid intake (mL day −1 ) 21.4 ± 2.4 24.9 ± 1.6** , ● 22.8 ± 2.8 22.4 ± 3.3 Total Se intake (µg day −1 ) 3.49 ± 0.29 3.35 ± 0.08 ●●● 6.38 ± 0.73*** 6.48 ± 0.79*** SKM weight (g) 0.99 ± 0.04 0.77 ± 0.06*** , ●● 0.95 ± 0.12 0.89 ± 0.07* Proteins in SKM (mg g −1 wet tissue) 43.3 ± 3.4 37.6 ± 4.3** , ●●● 41.6 ± 2.8 ●● 47.2 ± 4.7 CPK (UI L −1 ) 2215 ± 122 2292 ± 272 2095 ± 285 2481 ± 393 Creatinine (mg dL −1 ) 0.45 ± 0.03 0.47 ± 0.03 0.44 ± 0.06 0.51 ± 0.09 Pancreas weight (g) 1.01 ± 0.09 1.06 ± 0.07 1.11 ± 0.09 1.04 ± 0.13 Proteins in pancreas (mg g −1 wet tissue) 159 ± 35 284 ± 57*** 161 ± 31 ●●● 285 ± 28*** CPK: creatine phosphokinase. The results are expressed as mean ± SD and analyzed through two-way ANOVA, followed by Tukey’s test. The number of animals in each group is 8. Groups: C: control group, BD: binge drinking group, CSe: control selenium group, and BDSe: binge drinking selenium group. Statistic difference between groups is expressed as p-value: vs. C: *p< 0.05, **p< 0.01, ***p< 0.001; vs. BDSe: ● p< 0.05, ●● p< 0.01, ●●● p< 0.001. Paper Food & Function 7992 |Food Funct.,2024,15,7988–8007 This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 10 July 2024. Downloaded on 2/26/2025 7:44:28 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online animals. In addition, CSe rats had lower pAMPKα(p< 0.05), SIRT-1 (p< 0.001), NF-κB p65 (p< 0.001), atrogin-1/MAFbx (p< 0.01) and ULK1 (p< 0.01) expressions than BDSe rats; and slightly increased expressions of tAMPKα, pAMPKαand SIRT-1 (p< 0.05) against C animals. In Fig. 4, BD depleted the expressions of all selenoproteins in SKM as compared to C rats: GPx1 (p< 0.001), GPx4 (p< 0.001), SelM (p< 0.001), and SelP (p< 0.001), and the expression of SelP’s receptor LRP1 (p< 0.001). Conversely, the expressions of caspase-3 (p< 0.001) and pelF2α(p< 0.001) Fig. 1 Skeletal muscle oxidative balance in adolescent rats after binge drinking exposure and selenium supplementation. Superoxide dismutase (SOD) activity (A); catalase (CAT) activity (B); glutathione peroxidase (GPx) activity (C); malondialdehyde levels (MDA), showing lipid peroxidation (D); carbonyl group (CG), showing protein oxidation (E); NADPH oxidase 4 (NOX4) western blot expression and image with GAPDH as load control (F); and hydrogen peroxide (H 2 O 2 ) concentration, a reactive oxygen species (ROS) (G). The results are expressed as mean ± SD and analyzed through two-way ANOVA, followed by Tukey’s test. The number of animals in each group is 8. Groups: C: control group, BD: binge drinking group, CSe: control selenium group, and BDSe: binge drinking selenium group. Statistical difference between groups is expressed as p-value: vs. C: *p< 0.05, **p< 0.01, ***p< 0.001; vs. BDSe: ● p< 0.05, ●● p< 0.01, ●●● p< 0.001. Food & Function Paper This journal is © The Royal Society of Chemistry 2024 Food Funct.,2024,15,7988–8007 | 7993 Open Access Article. Published on 10 July 2024. Downloaded on 2/26/2025 7:44:28 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online were elevated in the BD rats. After Se supplementation to BD rats, the expression of selenoproteins significantly increased (GPx p< 0.001; GPx4 p< 0.001; SelM p< 0.01; and SelP p< 0.01), including LRP1 (p< 0.001); while caspase-3 and pelF2αwere reduced (p< 0.01 and p< 0.001, respectively). BDSe rats vs. C animals also presented higher levels of GPx1 (p< 0.05), GPx4 (p< 0.01) and caspase-3 (p < 0.01), and lower of SelP (p< 0.01). As regards the CSe group, GPx1 (p< 0.01) and GPx4 (p< 0.001) expressions were higher with respect to C. Of note, in the CSe rats, GPx4 and SelP expressions were elevated (p< 0.05) vs. BDSe, while caspase-3 was reduced (p< 0.001). When the BD group is compared to the C one, it increased serum IL-6 levels (p< 0.01); however, it decreased the secretion of several myokines: FGF21 (p< 0.01), irisin (p< 0.001), BDNF (p< 0.05), CX3CL1 (p< 0.001) and FSTL-1 (p< 0.01) (Fig. 5). When BD rats were supplemented with Se, compared to BD rats they presented higher serum levels of FGF21 (p< 0.05), irisin (p< 0.001), BDNF (p< 0.01), IL-15 (p< 0.01), CX3CL1 (p < 0.01) and FSTL-1 (p< 0.01), and lower levels of FABP-3 (p< Fig. 2 Expression of proteins related to protein synthesis in the skeletal muscle of adolescent rats after binge drinking exposure and selenium supplementation. Insulin receptor substrate 1 (IRS-1) (A); Akt1 (B); mammalian target of rapamycin 1 (mTOR) (C); phospho-mTOR (Ser2448) (pmTOR) (D); insulin-like growth factor 1 (IGF-1) (E); and western blot expression images with GAPDH as load control (F). The results are expressed as mean ± SD and analyzed through two-way ANOVA, followed by Tukey’s test. The number of animals in each group is 8. Groups: C: control group, BD: binge drinking group, CSe: control selenium group, and BDSe: binge drinking selenium group. Statistical difference between groups is expressed as p-value: vs. C: *p< 0.05, **p< 0.01, ***p< 0.001; vs. BDSe: ● p< 0.05, ●● p< 0.01, ●●● p< 0.001. Paper Food & Function 7994 |Food Funct.,2024,15,7988–8007 This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 10 July 2024. Downloaded on 2/26/2025 7:44:28 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Fig. 3 Expression of proteins related to proteolysis in the skeletal muscle of adolescent rats after binge drinking exposure and selenium supplementation. Total AMP-activated protein kinase α(AMPKα) (A); phospho-AMPKα(Thr172) (pAMPKα) (B); NAD + -dependent deacetylase sirtuin 1 (SIRT-1) (C); ratio tAMPK/pAMPK (D); nuclear factor kappa B (NF-κB p65) (E); muscle atrophy F-box (atrogin-1/MAFbx) (F); Unc-51-like autophagy activating kinase 1 (ULK1) (G); western blot expression images with GAPDH as load control (H) and serum myokine myostatin concentration (I). The results are expressed as mean ± SD and analyzed through two-way ANOVA, followed by Tukey’s test. The number of animals in each group is 8. Groups: C: control group, BD: binge drinking group, CSe: control selenium group, and BDSe: binge drinking selenium group. Statistical difference between groups is expressed as p-value: vs. C: *p< 0.05, **p< 0.01, ***p< 0.001; vs. BDSe: ● p< 0.05, ●● p< 0.01, ●●● p< 0.001. Food & Function Paper This journal is © The Royal Society of Chemistry 2024 Food Funct.,2024,15,7988–8007 | 7995 Open Access Article. Published on 10 July 2024. Downloaded on 2/26/2025 7:44:28 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Fig. 4 Selenoprotein expression in the skeletal muscle of adolescent rats after binge drinking exposure and selenium supplementation. Western blot expression images with GAPDH as load control (A); glutathione peroxidase 1 (GPx1) (B); glutathione peroxidase 4 (GPx4) (C); cleaved caspase-3 (D); selenoprotein M (SelM) (E); phospho-eukaryotic initiation factor 2α(Ser51) (pelF2α) (F); selenoprotein P (SelP) (G); and low-density lipoprotein receptor-related protein 1 (LRP1) (H). The results are expressed as mean ± SD and analyzed through two-way ANOVA, followed by Tukey’s test. The number of animals in each group is 8. Groups: C: control group, BD: binge drinking group, CSe: control selenium group, and BDSe: binge drinking selenium group. Statistical difference between groups is expressed as p-value: vs. C: *p< 0.05, **p< 0.01, ***p< 0.001; vs. BDSe: ● p< 0.05, ●● p< 0.01, ●●● p< 0.001. Paper Food & Function 7996 |Food Funct.,2024,15,7988–8007 This journal is © The Royal Society of Chemistry 2024 Open Access Article. Published on 10 July 2024. Downloaded on 2/26/2025 7:44:28 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online also affected insulin secretion (Fig. 6G). In a previous recent paper, 8 we confirmed that BD exposure during adolescence led to hyperglycemia and hyperinsulinemia, which were compatible with a general IR process. BD altered all antioxidant enzyme activities (Fig. 6), which resulted in lipid peroxidation in the pancreas. However, pancreatic β-cells, being especially sensitive to OS, 83 were apparently functional since serum insulin levels were increased. This could be due to the protective action of the energy sensor SIRT-1, overexpressed in our BD rats. SIRT-1 is also involved in the prevention of inflammatory and oxidative processes, being essential for the formation and maintenance of pancreatic β-cells. 84–87 Therefore, the impaired SKM insulin function found could be due to a disruption of the insulin signaling cascade in this tissue, and not to reduced pancreatic insulin secretion. Se supplementation to BD rats avoided lipid peroxidation by increasing the activity of the selenoprotein GPx and the antioxidant CAT. This enhanced CAT activity is important since this enzyme is crucial to eliminating H 2 O 2 in the pancreas. 88,89 BDSe rats also presented high SIRT-1 levels in the pancreas, collaborating with an appropriate pancreatic function; therefore, serum insulin levels were not affected in this group of rats. This fact and the proper SKM IRS-1 expression shown (Fig. 2A), indicate that the insulin signaling pathway is functioning properly and that protein biosynthesis is being actively maintained by this route, counteracting BDexposure effects. Eventually, CS rats presented higher activities of the three antioxidant enzymes; these increases were proportional, avoiding lipid peroxidation. It has been shown that an increase in the three enzymes and their combined action is very effective in stimulating the function of pancreatic β-cells. 90 Therefore, serum insulin levels were greatly increased; however, since IRS-1 expression in SKM and in other insulin target tissues of rats exposed to the same amount of selenite such as the liver were increased, and serum glucose levels were unaffected, 5 a general IR process is not taking place in CSe rats. Nevertheless, it is quite clear that Se is a potent element to induce insulin secretion and enhance insulin response to consider when required. 5. Conclusions Based on the results obtained, it has been confirmed that BD exposure during adolescence leads to oxidation and catabolism in SKM and the pancreas. Despite the increased serum insulin levels, its anabolic effects on SKM were decreased. Interestingly, it has been established for the first time that BD exposure during adolescence specifically decreases the expression of selenoproteins in SKM (GPx1, GPx4, SelP and SelM). This points to SKM as an important tissue in the SelP cycle after EtOH insults, since LRP1 expression was clearly decreased in BD rats, and links Se status to SKM development. Therefore, the selenite supplementation used in BD-exposed adolescent rats enhanced LRP1 and expression of selenoproteins, thereby improving SKM functions by modulating oxidative balance, energy-status, proteostasis, inflammation, apoptosis and ER homeostasis (Fig. 7). Besides, selenite not only adjusted the serum myokine profile toward a more anabolic one in SKM, but Se also had beneficial endocrine repercussions in the pancreas, WAT, liver, heart and brain, which positions it as a broad-spectrum therapy against alcohol consumption during adolescence. Furthermore, Se also decreased lipid peroxidation in the pancreas, contributing to normal serum insulin secretion. Data availability Data are available as ESI for review purposes (original western blot images). The rest of the data are all presented in this article. Conflicts of interest On behalf of all authors, the corresponding author states that there is no conflict of interest. Acknowledgements This research was funded by grants from the Andalusian Regional Government, which support the CTS-193 research group (2021/CTS-193; 2019/CTS-193), as well as by VII Plan Propio de Investigación y Transferencia –University of Seville 2022. 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