Arginine and Lysine Supplementation Potentiates the Beneficial ß-Hydroxy ß-Methyl Butyrate (HMB) Effects on Skeletal Muscle in a Rat Model of Diabetes.

Skeletal muscle is the key tissue for maintaining protein and glucose homeostasis, having a profound impact on the development of diabetes. Diabetes causes deleterious changes in terms of loss of muscle mass, which will contribute to reduced glucose uptake and therefore progression of the disease. Nutritional approaches in diabetes have been directed to increase muscle glucose uptake, and improving protein turnover has been at least partially an oversight. In muscle, ß-hydroxy ß-methyl butyrate (HMB) promotes net protein synthesis, while arginine and lysine increase glucose uptake, albeit their effects on promoting protein synthesis are limited. This study evaluates if the combination of HMB, lysine, and arginine could prevent the loss of muscle mass and function, reducing the progression of diabetes. Therefore, the combination of these ingredients was tested in vitro and in vivo. In muscle cell cultures, the supplementation enhances glucose uptake and net protein synthesis due to an increase in the amount of GLUT4 transporter and stimulation of the insulin-dependent signaling pathway involving IRS-1 and Akt. In vivo, using a rat model of diabetes, the supplementation increases lean body mass and insulin sensitivity and decreases blood glucose and serum glycosylated hemoglobin. In treated animals, an increase in GLUT4, creatine kinase, and Akt phosphorylation was detected, demonstrating the synergic effects of the three ingredients. Our findings showed that nutritional formulations based on the combination of HMB, lysine, and arginine are effective, not only to control blood glucose levels but also to prevent skeletal muscle atrophy associated with the progression of diabetes.

A Low Cost and Eco-Sustainable Device to Determine the End of the Disinfection Process in SODIS.

The lack of safe drinking water is one of the main health problems in many regions of the world. In order to face it, Solar water disinfection (SODIS) proposes the use of transparent plastic containers, which are filled with contaminated water, and exposed to direct sunlight until enough UV radiation is received to inactivate the pathogens. However, a reliable method for determining the end of the disinfection process is needed. Although several approaches have been proposed in the literature for this purpose, they do not strictly accomplish two critical constraints that are essential in this type of project, namely, low cost and sustainability. In this paper, we propose an electronic device to determine when the lethal UV dose has been reached in SODIS containers, which accomplishes both constraints mentioned above: on the one hand, its manufacturing cost is around EUR 12, which is much lower than the price of other electronic solutions; on the other hand, the device is sufficiently autonomous to work for months with small low-cost disposable batteries, thereby avoiding the use of rechargeable batteries, which are considered hazardous waste at the end of their useful life. In our approach, we first analyze different low cost UV sensors in order to select the most accurate one by comparing their response with a reference pattern provided by a radiometer. Then, an electronic device is designed using this sensor, which measures the accumulated UV radiation and compares this value with the lethal UV dose to determine the end of the disinfection process. Finally, the device has been manufactured and tested in real conditions to analyze its accuracy, obtaining satisfactory results.

Intake of slow-digesting carbohydrates is related to changes in the microbiome and its functional pathways in growing rats with obesity induced by diet.

Introduction: The main cause of insulin resistance in childhood is obesity, which contributes to future comorbidities as in adults. Although high-calorie diets and lack of exercise contribute to metabolic disease development, food quality rather than the quantity of macronutrients is more important than food density. The purpose of the present study was to examine the effects of changing the quality of carbohydrates from rapidly to slowly digestible carbohydrates on the composition of the gut microbiota and the profiles of the functional pathways in growing rats with obesity due to a high-fat diet (HFD). Methods: During the course of 4 weeks, rats growing on an HFD-containing carbohydrates with different digestive rates were fed either HFD-containing carbohydrates with a rapid digestion rate (OBE group) or HFD-containing carbohydrates with a slow digestion rate (OBE-ISR group). A non-obese group (NOB) was included as a reference, and rats were fed on a rodent standard diet (AIN93G). An analysis of gut microbiota was conducted using 16S rRNA-based metagenomics; a linear mixed-effects model (LMM) was used to determine changes in abundance between baseline and 4 weeks of treatment, and functional pathways were identified. Gut microbiota composition at bacterial diversity and relative abundance, at phylum and genus levels, and functional profiles were analyzed by integrating the Integrated Microbial Genomes (IMG) database. Results: The groups showed comparable gut microbiota at baseline. At the end of the treatment, animals from the ISR group exhibited differences at the phylum levels by decreasing the diversity of Fisher's index and Firmicutes (newly named as Bacillota), and increasing the Pielou's evenness and Bacteroidetes (newly named as Bacteroidota); at the genus level by increasing Alistipes, Bifidobacterium, Bacteroides, Butyricimonas, Lachnoclostridium, Flavonifractor, Ruminiclostridium 5, and Faecalibaculum and decreasing Muribaculum, Blautia, and Ruminiclostridium 9. Remarkably, relative abundances of genera Tyzzerella and Angelakisella were higher in the OBE group compared to NOB and OBE-ISR groups. In addition, some microbiota carbohydrate metabolism pathways such as glycolysis, glucuronic acid degradation, pentose phosphate pathway, methanogenesis, and fatty acid biosynthesis exhibited increased activity in the OBE-ISR group after the treatment. Higher levels of acetate and propionate were found in the feces of the ISR group compared with the NOB and OBE groups. Conclusion: The results of this study demonstrate that replacing rapidly digestible carbohydrates with slowly digestible carbohydrates within an HFD improve the composition of the gut microbiota. Consequently, metabolic disturbances associated with obesity may be prevented.

Quality More Than Quantity: The Use of Carbohydrates in High-Fat Diets to Tackle Obesity in Growing Rats.

Childhood obesity prevention is important to avoid obesity and its comorbidities into adulthood. Although the energy density of food has been considered a main obesogenic factor, a focus on food quality rather that the quantity of the different macronutrients is needed. Therefore, this study investigates the effects of changing the quality of carbohydrates from rapidly to slowly digestible carbohydrates on metabolic abnormalities and its impact on obesity in growing rats fed a high-fat diet (HFD). Growing rats were fed on HFD containing carbohydrates with different digestion rates: a HFD containing rapid-digesting carbohydrates (OBE group) or slow-digesting carbohydrates (ISR group), for 4 weeks and the effect on the metabolism and signaling pathways were analyzed in different tissues. Animals from OBE group presented an overweight/obese phenotype with a higher body weight gain and greater accumulation of fat in adipose tissue and liver. This state was associated with an increase of HOMA index, serum diacylglycerols and triacylglycerides, insulin, leptin, and pro-inflammatory cytokines. In contrast, the change of carbohydrate profile in the diet to one based on slow digestible prevented the obesity-related adverse effects. In adipose tissue, GLUT4 was increased and UCPs and PPARγ were decreased in ISR group respect to OBE group. In liver, GLUT2, FAS, and SRBP1 were lower in ISR group than OBE group. In muscle, an increase of glycogen, GLUT4, AMPK, and Akt were observed in comparison to OBE group. In conclusion, this study demonstrates that the replacement of rapidly digestible carbohydrates for slowly digestible carbohydrates within a high-fat diet promoted a protective effect against the development of obesity and its associated comorbidities.

Dietary Complex and Slow Digestive Carbohydrates Promote Bone Mass and Improve Bone Microarchitecture during Catch-Up Growth in Rats.

Catch-up growth is a process that promotes weight and height gains to recover normal growth patterns after a transient period of growth inhibition. Accelerated infant growth is associated with reduced bone mass and quality characterized by poor bone mineral density (BMD), content (BMC), and impaired microarchitecture. The present study evaluated the effects of a diet containing slow (SDC) or rapid (RDC) digestible carbohydrates on bone quality parameters during the catch-up growth period in a model of diet-induced stunted rats. The food restriction period negatively impacted BMD, BMC, and microarchitecture of appendicular and axial bones. The SDC diet was shown to improve BMD and BMC of appendicular and axial bones after a four-week refeeding period in comparison with the RDC diet. In the same line, the micro-CT analysis revealed that the trabecular microarchitecture of tibiae and vertebrae was positively impacted by the dietary intervention with SDC compared to RDC. Furthermore, features of the cortical microstructure of vertebra bones were also improved in the SDC group animals. Similarly, animals allocated to the SDC diet displayed modest improvements in growth plate thickness, surface, and volume compared to the RDC group. Diets containing the described SDC blend might contribute to an adequate bone formation during catch-up growth thus increasing peak bone mass, which could be linked to reduced fracture risk later in life in individuals undergoing transient undernutrition during early life.

Solar disinfection - An appropriate water treatment method to inactivate faecal bacteria in cold climates.

Solar disinfection (SODIS) is an inexpensive drinking water treatment method applied in tropical and sub-tropical low-income countries. However, it has been unclear whether it functions adequately also in colder climates. To investigate this issue, SODIS experiments were performed in the humid continental climate of Finland by exposing faecally contaminated drinking water to natural solar radiation at different water temperatures (8-23 °C) and UV intensities (12-19 W/m2) in polyethylene (PE) bags. To establish an adequate benchmark, SODIS experiments with the same experimental design were additionally conducted in the Mediterranean climate of Spain in typical conditions of SODIS application (~39 °C and 42 W/m2). Out of all experiments, the highest coliform and enterococci inactivation efficiencies in terms of lowest required doses for 4-log disinfection (25 Wh/m2 and 60 Wh/m2, respectively) were obtained in humid continental climate at the lowest studied mean water temperature (8-11 °C). Despite the low mean UV irradiance (~19 Wh/m2), 4-log disinfection of coliforms and enterococci were also reached fast in these conditions (1 h 27 min and 3 h 18 min, respectively). Overall, the doses required for disinfection increased as the water temperatures and UV intensities of the experiments rose. Disinfection of 4-logs (> 99.99%) of both bacteria was reached in all SODIS experiments within 6 h, suggesting SODIS could be a sufficient household water treatment method also in colder climates, unlike previously thought. The effects of different water temperatures on bacterial inactivation were also tested in the absence of sunlight. Together the obtained results indicate that while water temperatures below or close to the optima of coliforms and enterococci (~10 °C) alone do not cause inactivation, these temperatures may enhance SODIS performance. This phenomenon is attributed to slower bacterial metabolism and hence slower photorepair induced by the low water temperature.

Post-treatment of real municipal wastewater effluents by means of granular activated carbon (GAC) based catalytic processes: A focus on abatement of pharmaceutically active compounds.

Pharmaceutically active compounds (PhACs) widely present in urban wastewater effluents pose a threat to ecosystems in the receiving aquatic environment. In this work, efficiency of granular activated carbon (GAC) - based catalytic processes, namely catalytic wet peroxide oxidation (CWPO), peroxymonosulfate oxidation (PMS/GAC) and peroxydisulfate oxidation (PDS/GAC) at ambient temperature and pressure were studied for removal of 22 PhACs (ng L-1 level) that were present in secondary effluents of real urban wastewater. Concentrations of PhACs were measured using Ultra Performance Liquid Chromatography - Triple Quadrupole Mass Spectrometry (UPLC-QqQ-MS/MS). Catalytic experiments were conducted in discontinuous mode using up-flow fixed bed reactors with granular activated carbon (GAC) as a catalyst. The catalyst was characterized by means of N2 adsorption-desorption isotherm, mercury intrusion porosimetry (MIP), elemental analysis, X-ray fluorescence spectroscopy (WDXRF), X-ray diffraction (XRD), thermal gravimetry and differential temperature analyses coupled mass spectrometry (TGA-DTA-MS). Results indicate that the highest efficiency in terms of TOC removal was achieved during CWPO performed at optimal operational conditions (stoichiometric dose of H2O2; TOC removal ~ 82%) followed by PMS/GAC (initial PMS concentration 100 mg L-1; TOC removal ~73.7%) and PDS/GAC (initial PDS concentration 100 mg L-1; TOC removal ~ 67.9%) after 5 min of contact time. Full consumption of oxidants was observed in all cases for CWPO and PDS/GAC at contact times of 2.5 min, while for PMS/GAC it was 1.5 min. In general, for 18 out of 22 target PhACs, very high removal efficiencies (> 92%) were achieved in all tested processes (including adsorption) performed at optimal operational conditions during 5 min of contact time. However, moderate (40 - 70%) and poor (< 40%) removal efficiencies were achieved for salicylic acid, ofloxacin, norfloxacin and ciprofloxacin, which can be possibly attributed to insufficient contact time. Despite high efficiency of all studied processes for PhACs elimination from urban wastewater effluent, CWPO seems to be more promising for continuous operation.

Effect of the Combination of Superabsorbent Polymers for Autogenous Shrinkage Control with Steel Fibers of High-Performance Concrete under Uniaxial Tension Using DIC.

This paper presents a study of the effect of a superabsorbent polymer (SAP) for autogenous shrinkage control on the uniaxial tensile behavior of steel fiber reinforced concrete (SFRC). The use of fibers and SAP potentially increases the durability of the concrete, preventing cracking by autogenous shrinkage and enhancing post-cracking behavior. Furthermore, SAP can provide further hydration for self-healing purposes and improve the ductility of the SFRC. In order to evaluate the effect of the addition of SAP in SFRC, dog-bone SFRC specimens with different dosages of superabsorbent polymers were cast and tested under uniaxial tension. The digital image correlation (DIC) technique was used to understand the effect of SAP on the steel fibers' crack-bridging mechanisms. Surface strains and crack openings were inferred using the DIC technique. The effect of SAP and fibers on fresh and hardened concrete was individually investigated by flow tests and compressive strength tests. Autogenous shrinkage was measured in plain concrete to investigate the minimum SAP content required to mitigate autogenous shrinkage of 0.3%. The use of 0.3% SAP was also sufficient to reach multiple cracking behavior. This content of SAP completely suppressed the autogenous shrinkage with minimal side effects on compressive strength. An analytical formulation for the tensile behavior of SFRC was developed using the variable engagement model, presenting a mean correlation of R2 of 0.97 with the experimental results.

Dietary Complex and Slow Digestive Carbohydrates Prevent Fat Deposits During Catch-Up Growth in Rats.

A nutritional growth retardation study, which closely resembles the nutritional observations in children who consumed insufficient total energy to maintain normal growth, was conducted. In this study, a nutritional stress in weanling rats placed on restricted balanced diet for 4 weeks is produced, followed by a food recovery period of 4 weeks using two enriched diets that differ mainly in the slow (SDC) or fast (RDC) digestibility and complexity of their carbohydrates. After re-feeding with the RDC diet, animals showed the negative effects of an early caloric restriction: an increase in adiposity combined with poorer muscle performance, insulin resistance and, metabolic inflexibility. These effects were avoided by the SDC diet, as was evidenced by a lower adiposity associated with a decrease in fatty acid synthase expression in adipose tissue. The improved muscle performance of the SDC group was based on an increase in myocyte enhancer factor 2D (MEF2D) and creatine kinase as markers of muscle differentiation as well as better insulin sensitivity, enhanced glucose uptake, and increased metabolic flexibility. In the liver, the SDC diet promoted glycogen storage and decreased fatty acid synthesis. Therefore, the SDC diet prevents the catch-up fat phenotype through synergistic metabolic adaptations in adipose tissue, muscle, and liver. These coordinated adaptations lead to better muscle performance and a decrease in the fat/lean ratio in animals, which could prevent long-term negative metabolic alterations such as obesity, insulin resistance, dyslipidemia, and liver fat deposits later in life.

Programming Skeletal Muscle Metabolic Flexibility in Offspring of Male Rats in Response to Maternal Consumption of Slow Digesting Carbohydrates during Pregnancy.

Skeletal muscle plays a relevant role in metabolic flexibility and fuel usage and the associated muscle metabolic inflexibility due to high-fat diets contributing to obesity and type 2 diabetes. Previous research from our group indicates that a high-fat and rapid-digesting carbohydrate diet during pregnancy promotes an excessive adipogenesis and also increases the risk of non-alcoholic fatty liver disease in the offspring. This effect can be counteracted by diets containing carbohydrates with similar glycemic load but lower digestion rates. To address the role of the skeletal muscle in these experimental settings, pregnant rats were fed high-fat diets containing carbohydrates with similar glycemic load but different digestion rates, a high fat containing rapid-digesting carbohydrates diet (HF/RD diet) or a high fat containing slow-digesting carbohydrates diet (HF/SD diet). After weaning, male offspring were fed a standard diet for 3 weeks (weaning) or 10 weeks (adolescence) and the impact of the maternal HF/RD and HF/SD diets on the metabolism, signaling pathways and muscle transcriptome was analyzed. The HF/SD offspring displayed better muscle features compared with the HF/RD group, showing a higher muscle mass, myosin content and differentiation markers that translated into a greater grip strength. In the HF/SD group, metabolic changes such as a higher expression of fatty acids (FAT/CD36) and glucose (GLUT4) transporters, an enhanced glycogen content, as well as changes in regulatory enzymes such as muscle pyruvate kinase and pyruvate dehydrogenase kinase 4 were found, supporting an increased muscle metabolic flexibility and improved muscle performance. The analysis of signaling pathways was consistent with a better insulin sensitivity in the muscle of the HF/SD group. Furthermore, increased expression of genes involved in pathways leading to muscle differentiation, muscle mass regulation, extracellular matrix content and insulin sensitivity were detected in the HF/SD group when compared with HF/RD animals. In the HF/SD group, the upregulation of the ElaV1/HuR gene could be one of the main regulators in the positive effects of the diet in early programming on the offspring. The long-lasting programming effects of the HF/SD diet during pregnancy may depend on a coordinated gene regulation, modulation of signaling pathways and metabolic flexibility that lead to an improved muscle functionality. The dietary early programming associated to HF/SD diet has synergic and positive crosstalk effects in several tissues, mainly muscle, liver and adipose tissue, contributing to maintain the whole body homeostasis in the offspring.

A Slow-Digesting Carbohydrate Diet during Rat Pregnancy Protects Offspring from Non-Alcoholic Fatty Liver Disease Risk through the Modulation of the Carbohydrate-Response Element and Sterol Regulatory Element Binding Proteins.

High-fat (HF) and rapid digestive (RD) carbohydrate diets during pregnancy promote excessive adipogenesis in offspring. This effect can be corrected by diets with similar glycemic loads, but low rates of carbohydrate digestion. However, the effects of these diets on metabolic programming in the livers of offspring, and the liver metabolism contributions to adipogenesis, remain to be addressed. In this study, pregnant insulin-resistant rats were fed high-fat diets with similar glycemic loads but different rates of carbohydrate digestion, High Fat-Rapid Digestive (HF-RD) diet or High Fat-Slow Digestive (HF-SD) diet. Offspring were fed a standard diet for 10 weeks, and the impact of these diets on the metabolic and signaling pathways involved in liver fat synthesis and storage of offspring were analyzed, including liver lipidomics, glycogen and carbohydrate and lipid metabolism key enzymes and signaling pathways. Livers from animals whose mothers were fed an HF-RD diet showed higher saturated triacylglycerol deposits with lower carbon numbers and double bond contents compared with the HF-SD group. Moreover, the HF-RD group exhibited enhanced glucose transporter 2, pyruvate kinase (PK), acetyl coenzyme A carboxylase (ACC) and fatty acid (FA) synthase expression, and a decrease in pyruvate carboxylase (PyC) expression leading to an altered liver lipid profile. These parameters were normalized in the HF-SD group. The changes in lipogenic enzyme expression were parallel to changes in AktPKB phosphorylation status and nuclear expression in carbohydrate-response element and sterol regulatory element binding proteins. In conclusion, an HF-RD diet during pregnancy translates to changes in liver signaling and metabolic pathways in offspring, enhancing liver lipid storage and synthesis, and therefore non-alcoholic fatty liver disease (NAFLD) risk. These changes can be corrected by feeding an HF-SD diet during pregnancy.

Feeding a slowly digestible carbohydrate diet during pregnancy of insulin-resistant rats prevents the excess of adipogenesis in their offspring.

An obesogenic environment during pregnancy has been shown to increase the risk of dysregulation on adipogenesis and insulin resistance in the offspring. Being essential for the growing fetus, glucose supply is guaranteed by a number of modifications in the mother's metabolism, and thus, glucose control during pregnancy especially among obese or diabetic women is paramount to prevent adverse consequences in their children. Besides the election of low-glycemic-index carbohydrates, the rate of carbohydrate digestion could be relevant to keep a good glucose control. In the present study, we compared the effects of two high-fat diets with similar glycemic load but different rates of carbohydrate digestion given to pregnant insulin-resistant rats. After birth, all animals were fed a standard diet until age 14 weeks. We analyzed offspring body composition, plasma and adipocyte lipidomics, lipid metabolism in adipose tissue and insulin sensitivity. Those animals whose mothers were fed the rapid-digesting carbohydrate diet exhibited an excessive adipogenesis. Thus, these animals showed a marked lipidemia, increased lipid synthesis in the adipose tissue and reduced glucose transporter amount in the adipose. On the contrary, those animals whose mothers were fed the slow-digesting carbohydrate diet showed a profile in the measured parameters closer to that of the offspring of healthy mothers. These results support the hypothesis that not only glycemic index but the rate of carbohydrate digestion during gestation may be critical to regulate the programming of adipogenesis in the offspring.

Combining sun-based technologies (microalgae and solar disinfection) for urban wastewater regeneration.

Solar disinfection (SODIS) of urban wastewater can be a suitable technology for improving the microbiological quality of reclaimed water as a complement to other extensive and environmentally friendly technologies such as microalgae biotreatment. The objective of this work is to evaluate the feasibility of incorporating the SODIS technology at the end of a pilot scale urban wastewater treatment plant (WWTP) where the processes are based on microalgae biotechnology and comprising three Upflow Anaerobic Sludge Blanket (UASB, 20m3 each one) reactor, six High Rate Algal Ponds (HRAP, 32m2 each one), and a Dissolved Air Flotation (DAF, 1m3) unit. E. coli concentration was monitored at the effluent of the different units (UASB, HRAP, DAF) of the pilot WWTP. The efficiency of the SODIS process was studied for the inactivation of three of the commonly employed indicator microorganisms (Escherichia coli, Enterococcus spp. and Clostridium perfringens) using a compound parabolic collector (CPC) for five months under various conditions of irradiance and temperature. E. coli and Enterococcus spp. were more effectively disinfected by the SODIS unit (2.9 and 2.5 logarithms of reduction on average, respectively) than by the HRAP (2 and 1.1) or the DAF (0.9 and 0.1). On the contrary, the DAF technology achieved better reduction rates of C. perfringens (1.7) than the SODIS (0.9) and the HRAP (0.1). No regrowth of any microorganisms was detected during dark storage after the SODIS treatment. Incorporating a SODIS unit after the non-conventional WWTP processes substantially increases the possibilities for reuse of the treated water after receiving a cumulative UV radiation dose of 25W·h/m2 (50min of normalized time of solar illumination). The surface requirement of the SODIS equipment would be 3.5 times smaller than the HRAP's surface.

A Rat Immobilization Model Based on Cage Volume Reduction: A Physiological Model for Bed Rest?

Bed rest has been an established treatment in the past prescribed for critically illness or convalescing patients, in order to preserve their body metabolic resource, to prevent serious complications and to support their rapid path to recovery. However, it has been reported that prolonged bed rest can have detrimental consequences that may delay or prevent the recovery from clinical illness. In order to study disuse-induced changes in muscle and bone, as observed during prolonged bed rest in humans, an innovative new model of muscle disuse for rodents is presented. Basically, the animals are confined to a reduced space designed to restrict their locomotion movements and allow them to drink and eat easily, without generating physical stress. The animals were immobilized for either 7, 14, or 28 days. The immobilization procedure induced a significant decrease of food intake, both at 14 and 28 days of immobilization. The reduced food intake was not a consequence of a stress condition induced by the model since plasma corticosterone levels -an indicator of a stress response- were not altered following the immobilization period. The animals showed a significant decrease in soleus muscle mass, grip force and cross-sectional area (a measure of fiber size), together with a decrease in bone mineral density. The present model may potentially serve to investigate the effects of bed-rest in pathological states characterized by a catabolic condition, such as diabetes or cancer.

Apple polyphenol extract improves insulin sensitivity in vitro and in vivo in animal models of insulin resistance.

BACKGROUND: Apple polyphenols could represent a novel nutritional approach in the management and control of blood glucose, especially in type 2 diabetics. The aim of this study was to test the therapeutic potential of an apple polyphenol extract (APE) in an insulin-resistant rat model and to determine the molecular basis of insulin sensitivity action in skeletal muscle cells. METHODS: Acute effect of APE on the postprandial hyperglycemic response was assayed in 15 week old obese Zucker rats (OZR), by using a meal tolerance test (MTT). The ability of APE to improve whole peripheral insulin sensitivity was also assayed in a chronic study by using the euglycemic-hyperinsulinemic clamp technique. To elucidate the molecular mechanisms, rat L6 myotubes were used. Glucose uptake was measured by using 2-[3H]-Deoxy-Glucose (2-DG) and specific inhibitors, as well as phosphorylation status of key kinases, were used to determine the implicated signaling pathway. RESULTS: In vivo study showed that nutritional intervention with APE induced an increase of insulin sensitivity with an increase of glucose infusion rate (GIR) of 45 %. Additionally, in vitro results showed a synergistic effect between APE and insulin as well as increased glucose uptake through GLUT4 translocation in muscle cells. This translocation was mediated by phosphatydil inositol 3-kinase (PI3K) and peroxisome proliferator-activated receptor-gamma (PPARγ) signaling pathways. CONCLUSIONS: As a whole, this study describes the mechanisms involved in the insulin sensitizing effect of APE, which could be considered a promising ingredient for inclusion in nutritional products focused on the management of chronic diseases such as diabetes.

Conversion of leucine to ß-hydroxy-ß-methylbutyrate by α-keto isocaproate dioxygenase is required for a potent stimulation of protein synthesis in L6 rat myotubes.

BACKGROUND: L-Leu and its metabolite ß-hydroxy-ß-methylbutyrate (HMB) stimulate muscle protein synthesis enhancing the phosphorylation of proteins that regulate anabolic signalling pathways. Alterations in these pathways are observed in many catabolic diseases, and HMB and L-Leu have proven their anabolic effects in in vivo and in vitro models. The aim of this study was to compare the anabolic effects of L-Leu and HMB in myotubes grown in the absence of any catabolic stimuli. METHODS: Studies were conducted in vitro using rat L6 myotubes under normal growth conditions (non-involving L-Leu-deprived conditions). Protein synthesis and mechanistic target of rapamycin signalling pathway were determined. RESULTS: Only HMB was able to increase protein synthesis through a mechanism that involves the phosphorylation of the mechanistic target of rapamycin as well as its downstream elements, pS6 kinase, 4E binding protein-1, and eIF4E. HMB was significantly more effective than L-Leu in promoting these effects through an activation of protein kinase B/Akt. Because the conversion of L-Leu to HMB is limited in muscle, L6 cells were transfected with a plasmid that codes for α-keto isocaproate dioxygenase, the key enzyme involved in the catabolic conversion of α-keto isocaproate into HMB. In these transfected cells, L-Leu was able to promote protein synthesis and mechanistic target of rapamycin regulated pathway activation equally to HMB. Additionally, these effects of leucine were reverted to a normal state by mesotrione, a specific inhibitor of α-keto isocaproate dioxygenase. CONCLUSION: Our results suggest that HMB is an active L-Leu metabolite able to maximize protein synthesis in skeletal muscle under conditions, in which no amino acid deprivation occurred. It may be proposed that supplementation with HMB may be very useful to stimulate protein synthesis in wasting conditions associated with chronic diseases, such as cancer or chronic heart failure.

Maternal Dietary Supplementation with Oligofructose-Enriched Inulin in Gestating/Lactating Rats Preserves Maternal Bone and Improves Bone Microarchitecture in Their Offspring.

Nutrition during pregnancy and lactation could exert a key role not only on maternal bone, but also could influence the skeletal development of the offspring. This study was performed in rats to assess the relationship between maternal dietary intake of prebiotic oligofructose-enriched inulin and its role in bone turnover during gestation and lactation, as well as its effect on offspring peak bone mass/architecture during early adulthood. Rat dams were fed either with standard rodent diet (CC group), calcium-fortified diet (Ca group), or prebiotic oligofructose-enriched inulin supplemented diet (Pre group), during the second half of gestation and lactation. Bone mineral density (BMD) and content (BMC), as well as micro-structure of dams and offspring at different stages were analysed. Dams in the Pre group had significantly higher trabecular thickness (Tb.Th), trabecular bone volume fraction (BV/TV) and smaller specific bone surface (BS/BV) of the tibia in comparison with CC dams. The Pre group offspring during early adulthood had an increase of the lumbar vertebra BMD when compared with offspring of CC and Ca groups. The Pre group offspring also showed significant increase versus CC in cancellous and cortical structural parameters of the lumbar vertebra 4 such as Tb.Th, cortical BMD and decreased BS/BV. The results indicate that oligofructose-enriched inulin supplementation can be considered as a plausible nutritional option for protecting against maternal bone loss during gestation and lactation preventing bone fragility and for optimizing peak bone mass and architecture of the offspring in order to increase bone strength.

ß-Hydroxy-ß-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells.

ß-Hydroxy-ß-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.

ß-Hydroxy-ß-methylbutyrate (HMB) normalizes dexamethasone-induced autophagy-lysosomal pathway in skeletal muscle.

Dexamethasone-induced muscle atrophy is due to an increase in protein breakdown and a decrease in protein synthesis, associated with an over-stimulation of the autophagy-lysosomal pathway. These effects are mediated by alterations in IGF-1 and PI3K/Akt signaling. In this study, we have investigated the effects of ß-Hydroxy-ß-methylbutyrate (HMB) on the regulation of autophagy and proteosomal systems. Rats were treated during 21 days with dexamethasone as a model of muscle atrophy. Co-administration of HMB attenuated the effects promoted by dexamethasone. HMB ameliorated the loss in body weight, lean mass and the reduction of the muscle fiber cross-sectional area (shrinkage) in gastrocnemius muscle. Consequently, HMB produced an improvement in muscle strength in the dexamethasone-treated rats. To elucidate the molecular mechanisms responsible for these effects, rat L6 myotubes were used. In these cells, HMB significantly attenuated lysosomal proteolysis induced by dexamethasone by normalizing the changes observed in autophagosome formation, LC3 II, p62 and Bnip3 expression after dexamethasone treatment. HMB effects were mediated by an increase in FoxO3a phosphorylation and concomitant decrease in FoxO transcriptional activity. The HMB effect was due to the restoration of Akt signaling diminished by dexamethasone treatment. Moreover, HMB was also involved in the regulation of the activity of ubiquitin and expression of MurF1 and Atrogin-1, components of the proteasome system that are activated or up-regulated by dexamethasone. In conclusion, in vivo and in vitro studies suggest that HMB exerts protective effects against dexamethasone-induced muscle atrophy by normalizing the Akt/FoxO axis that controls autophagy and ubiquitin proteolysis.

Salacia oblonga extract increases glucose transporter 4-mediated glucose uptake in L6 rat myotubes: role of mangiferin.

BACKGROUND AND AIMS: To evaluate if the antidiabetic properties of Salacia oblonga extract are mediated not only by inhibiting intestinal alpha-glycosidases but also by enhancing glucose transport in muscle and adipose cells. METHODS: S. oblonga extract effects on 2-deoxy-D-glucose uptake were assayed in muscle L6-myotubes and 3T3-adipocytes. In L6-myotubes, the amount and translocation of glucose transporters were assayed. A fractionation of the extract was carried out to identify the active compounds. Furthermore, we analyzed the phosphorylation status of key components of signaling pathways that are involved in the molecular mechanisms regulating glucose uptake. RESULTS: S. oblonga extract increased 2-deoxy-D-glucose uptake by 50% in L6-myotubes and 3T3-adipocytes. In L6-myotubes, the extract increased up to a 100% the GLUT4 content, activating GLUT4 promoter transcription and its translocation to the plasma membrane. Mangiferin was identified as the bioactive compound. Furthermore, mangiferin effects were concomitant with the phosphorylation of 5'-AMP-activated protein kinase without the activation of PKB/Akt. The effect of mangiferin on 2-deoxy-D-glucose uptake was blocked by GW9662, an irreversible PPAR-gamma antagonist. CONCLUSIONS: S. oblonga extract and mangiferin may exert their antidiabetic effect by increasing GLUT4 expression and translocation in muscle cells. These effects are probably mediated through two independent pathways that are related to 5'-AMP-activated protein kinase and PPAR-gamma.