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The prevalence of non-alcoholic fatty liver disease (NAFLD) and its severe form, non-alcoholic steatohepatitis (NASH), is higher in men than in women of reproductive age, and postmenopausal women are especially susceptible to developing the disease. Aim: we evaluated if female apolipoprotein E (ApoE) KO mice were protected against Western-diet (WD)-induced NASH. Methods: Female ovariectomized (OVX) ApoE KO mice or sham-operated (SHAM) mice were fed either a WD or a regular chow (RC) for 7 weeks. Additionally, OVX mice fed a WD were treated with either estradiol (OVX + E2) or vehicle (OVX). Results: Whole-body fat, plasma glucose, and plasma insulin were increased and associated with increased glucose intolerance in OVX mice fed a WD (OVX + WD). Plasma and hepatic triglycerides, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) hepatic enzymes were also increased in the plasma of OVX + WD group, which was associated with hepatic fibrosis and inflammation. Estradiol replacement in OVX mice reduced body weight, body fat, glycemia, and plasma insulin associated with reduced glucose intolerance. Treatment also reduced hepatic triglycerides, ALT, AST, hepatic fibrosis, and inflammation in OVX mice. Conclusions: These data support the hypothesis that estradiol protects OVX ApoE KO mice from NASH and glucose intolerance.

Introduction: Exercise is an important therapeutic strategy for preventing and treating myocardial infarction (MI)-induced cardiac remodeling and heart failure. However, the myocardial effects of resistance exercise on infarcted hearts are not completely established. In this study, we investigated the effects of resistance exercise on structural, functional, and molecular cardiac alterations in infarcted rats. Methods: Three months after MI induction or simulated surgery, Wistar rats were assigned into three groups: Sham (n = 14); MI (n = 9); and exercised MI (MI-Ex, n = 13). Exercised rats performed, 3 times a week for 12 weeks, four climbs on a ladder with progressive loads. Cardiac structure and left ventricle (LV) function were analyzed by echocardiogram. Myocyte diameters were evaluated in hematoxylin- and eosin-stained histological sections as the smallest distance between borders drawn across the nucleus. Myocardial energy metabolism, lipid hydroperoxide, malondialdehyde, protein carbonylation, and antioxidant enzyme activities were evaluated by spectrophotometry. Gene expressions of NADPH oxidase subunits were evaluated by RT-PCR. Statistical analyses were performed using ANOVA and Tukey or Kruskal–Wallis and Dunn’s test. Results: Mortality did not differ between the MI-Ex and MI groups. MI had dilated left atrium and LV, with LV systolic dysfunction. Exercise increased the maximum load-carrying capacity, with no changes in cardiac structure or LV function. Myocyte diameters were lower in MI than in Sham and MI-Ex. Lactate dehydrogenase and creatine kinase activity were lower in MI than in Sham. Citrate synthase and catalase activity were lower in MI and MI-Ex than in Sham. Lipid hydroperoxide concentration was lower in MI-Ex than in MI. Nox2 and p22phox gene expressions were higher in MI-Ex than in Sham. Gene expression of Nox4 was higher in MI and MI-Ex than in Sham, and p47phox was lower in MI than in Sham. Conclusion: Late resistance exercise was safe in infarcted rats. Resistance exercise improved maximum load-carrying capacity, reduced myocardial oxidative stress, and preserved myocardial metabolism, with no changes in cardiac structure or left ventricle function in infarcted rats.

In pathological cardiac hypertrophy, the heart is more dependent on glucose than fatty acids. This shift in energy metabolism occurs due to several factors, including the oxygen deficit, which activates hypoxia-inducible factor-1α (HIF-1α), a critical molecule related to glucose metabolism. However, there are gaps regarding the behavior of key proteins in the glycolytic pathway and HIF-1α during the transition from hypertrophy to heart failure (HF). This study assesses the hypothesis that there is an early change and enhancement of HIF-1α and the glycolytic pathway, as well as an association between them during cardiac remodeling. Sham and aortic stenosis Wistar rats were analyzed at 2, 6, and 18 weeks and in HF (n = 10–18). Cardiac structure and function were investigated by echocardiogram. Myocardial glycolysis, the aerobic and anaerobic pathways and glycogen were analyzed by enzymatic assay, Western blot, and enzyme-linked immunosorbent assay (ELISA). The following were observed: increased left ventricular hypertrophy; early diastolic function change and severe systolic and diastolic dysfunction in HF; increased HIF-1α in the 2nd week and in HF; precocious alteration and intensification of glycolysis with a shift to anaerobic metabolism from the 6th week onwards; association between HIF-1α, glycolysis, and the anaerobic pathway. Our hypothesis was confirmed as there was an early change and intensification in glucose metabolism, alteration in HIF-1α, and an association between data during the progression from hypertrophy to heart failure.

Sepsis is a life-threatening condition with high hospital mortality. Elevated mortality has also been observed in patients after hospital discharge due to post-sepsis syndrome (PSS). The etiology of PSS is still not entirely known, but it involves inflammation. Plasma extracellular vesicles (EVs) are recognized as a unique mechanism of intercellular communication in inflammatory processes. It has been reported that EV microRNA (miRNA) production during the acute sepsis phase may persist until after disease resolution and is associated with PSS. We employed mass spectrometry and qPCR analysis to determine the protein and miRNA composition of plasma-derived EVs of 36 patients during sepsis-related hospitalization, immediately after ICU discharge (post-sepsis), and three, six, twelve, and up to 36 months post-sepsis. We determined that patients’ immune system cells were the primary EV source. Fifteen differentially expressed EV miRNAs (DEmiRs) were identified in samples from septic patients compared to the control group. Predictive analyses revealed that these DEmiRs could influence inflammation by modulating pathways mediated by NF-κB, STAT3, and TLR4 signaling activation. Thirteen miRNAs (-15b-5p,-16-5p,-20a-5p,-25-3p,-27a-3p,-29a-3p,-30d-5p,-93-5p,-146a-5p,-148a-3p,-191-5p,-195-5p,-223-3p) were downregulated in the death group compared to the survivor group, making them candidate prognostic markers of ICU survival. One year after ICU discharge, the expression of miR-21-5p and miR-195-5p were decreased in the survivor group. The miRNAs identified in the present study represent potential biomarkers for the survival prognosis of post-sepsis patients.

New enzyme functions exist within the increasing number of unannotated protein sequences. Novel enzyme discovery is necessary to expand the pathways that can be accessed by metabolic engineering for the biosynthesis of functional compounds. Accordingly, various machine learning models have been developed to predict enzymatic reactions. However, the ability to predict unknown reactions that are not included in the training data has not been clarified. In order to cover uncertain and unknown reactions, a wider range of reaction types must be demonstrated by the models. Here, we establish 16 expanded enzymatic reaction prediction models developed using various machine learning algorithms, including deep neural network. Improvements in prediction performances over that of our previous study indicate that the updated methods are more effective for the prediction of enzymatic reactions. Overall, the deep neural network model trained with combined substrate-enzyme-product information exhibits the highest prediction accuracy with Macro F

Although current guidelines recommend resistance exercise in combination with aerobic training to increase muscle strength and prevent skeletal muscle loss during cardiac remodeling, its effects are not clear. In this study, we evaluated the effects of resistance training on cardiac remodeling and the soleus muscle in long-term myocardial infarction (MI) rats. Methods: Three months after MI induction, male Wistar rats were assigned to Sham (n = 14), MI (n = 9), and resistance exercised MI (R-MI, n = 13) groups. The rats trained three times a week for 12 weeks on a climbing ladder. An echocardiogram was performed before and after training. Protein expression of the insulin-like growth factor (IGF)-1/protein kinase B (Akt)/rapamycin target complex (mTOR) pathway was analyzed by Western blot. Results: Mortality rate was higher in MI than Sham; in the R-MI group, mortality rate was between that in MI and Sham and did not differ significantly from either group. Exercise increased maximal load capacity without changing cardiac structure and left ventricular function in infarcted rats. Infarction size did not differ between infarcted groups. Catalase activity was lower in MI than Sham and glutathione peroxidase lower in MI than Sham and R-MI. Protein expression of p70S6K was lower in MI than Sham and p-FoxO3 was lower in MI than Sham and R-MI. Energy metabolism did not differ between groups, except for higher phosphofrutokinase activity in R-MI than MI. Conclusion: Resistance exercise is safe and increases muscle strength regardless structural and functional cardiac changes in myocardial-infarcted rats. This exercise modality attenuates soleus glycolytic metabolism changes and improves the expression of proteins required for protein turnover and antioxidant response.

In the present study we investigated the participation of hepatic peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) in the metabolic programming of newborn rats exposed in utero to dexamethasone (DEX). On the 21st day of life, fasted offspring born to DEX-treated mothers displayed increased conversion of pyruvate into glucose with simultaneous upregulation of PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (glucose-6-phosphatase). Increased oxidative phosphorylation, higher ATP/ADP ratio and mitochondrial biogenesis and lower pyruvate levels were also found in the progeny of DEX-treated mothers. On the other hand, the 21-day-old progeny of DEX-treated mothers had increased hepatic triglycerides (TAG) and lower CPT-1 activity when subjected to short-term fasting. At the mechanistic level, rats exposed in utero to DEX exhibited increased hepatic PGC-1α protein content with lower miR-29a-c expression. Increased PGC-1α content was concurrent with increased association to HNF-4α and NRF1 and reduced PPARα expression. The data presented herein reveal that changes in the transcription machinery in neonatal liver of rats born to DEX-treated mothers leads to an inflexible metabolic response to fasting. Such programming is hallmarked by increased oxidative phosphorylation of pyruvate with impaired FFA oxidation and hepatic TAG accumulation.

Introduction Resistance exercise (RE) provides several benefits for healthy individuals. However, its effects during heart failure are unclear. The aim of this study was to evaluate the effects of RE on functional capacity, cardiac remodeling, and soleus muscle molecular and biochemical features in rats with myocardial infarction (MI). Methods Three months after MI or simulated surgery (Sham), Wistar rats were divided into three groups: Sham (n=14), MI (n=13), and MI subjected to RE (RE-MI, n=14). Exercised rats trained 3 times a week performing four climbs with progressive loads on a ladder over 12 weeks. Functional capacity was assessed by maximum carrying capacity test in the ladder and exercise tolerance test in treadmill. Echocardiogram was performed at the end of the study. Energy metabolism and antioxidant enzyme activities were assessed by spectrophotometry in the soleus muscle. Oxidative stress markers were analyzed in soleus muscle (lipid hydroperoxide) or serum (malondialdehyde and protein carbonylation). Protein expression of insulin type-1 growth factor-like pathway, protein kinase B, and rapamycin target complex was analyzed by Western-blot. Statistical analysis: ANOVA and Bonferroni or Dunn, Student's t and Goodman tests; p Results Mortality was higher in MI than Sham. Infarction size did not differ between groups. Resistance exercise increased maximum load carrying capacity, without changing functional capacity or cardiac remodeling. Catalase activity was lower in MI than Sham and glutathione peroxidase activity was lower in MI than Sham and RE-MI. Protein carbonylation was higher in RE-MI than MI. Energy metabolism did not differ between groups, except for lower phosphofructokinase activity in RE-MI than MI. Expression of p70s6K, p-FoxO3a, and p-FoxO3a-to-FoxO3a ratio was lower, and p-p70s6K-to-p70s6K ratio was higher in MI than Sham. Conclusion The practice of resistance exercise is safe, attenuates mortality, and improves maximum load carrying capacity regardless of changes in cardiac remodeling in infarcted rats. In soleus muscle, resistance exercise preserves phosphofructokinase and antioxidant enzyme activity and expression of the proteins involved in muscle trophism. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): CNPq UNESP

Flux balance analysis (FBA) using genome-scale metabolic model (GSM) is a useful method for improving the bio-production of useful compounds. However, FBA often does not impose important constraints such as nutrients uptakes, by-products excretions and gases (oxygen and carbon dioxide) transfers. Furthermore, important information on metabolic engineering such as enzyme amounts, activities, and characteristics caused by gene expression and enzyme sequences is basically not included in GSM. Therefore, simple FBA is often not sufficient to search for metabolic manipulation strategies that are useful for improving the production of target compounds. In this study, we proposed a method using literature and enzyme search to complement the FBA-based metabolic manipulation strategies. As a case study, this method was applied to shikimic acid production by Corynebacterium glutamicum to verify its usefulness. As unique strategies in literature-mining, overexpression of the transcriptional regulator SugR and gene disruption related to by-products productions were complemented. In the search for alternative enzyme sequences, it was suggested that those candidates are searched for from various species based on features captured by deep learning, which are not simply homologous to amino acid sequences of the base enzymes.

The role of microRNAs in metabolic diseases has been recognized and modulation of them could be a promising strategy to treat obesity and obesity-related diseases. The major purpose of this study was to test the hypothesis that intramuscular miR-1 precursor replacement therapy could improve metabolic parameters of mice fed a high-fat diet. To this end, we first injected miR-1 precursor intramuscularly in high-fat diet-fed mice and evaluated glucose tolerance, insulin sensitivity, and adiposity. miR-1-treated mice did not lose weight but had improved insulin sensitivity measured by insulin tolerance test. Next, using an in vitro model of insulin resistance by treating C2C12 cells with palmitic acid (PA), we overexpressed miR-1 and measured p-Akt content and the transcription levels of a protein related to fatty acid oxidation. We found that miR-1 could not restore insulin sensitivity in C2C12 cells, as indicated by p-Akt levels and that miR-1 increased expression of Pgc1a and Cpt1b in PA-treated cells, suggesting a possible role of miR-1 in mitochondrial respiration. Finally, we analyzed mitochondrial oxygen consumption in primary skeletal muscle cells treated with PA and transfected with or without miR-1 mimic. PA-treated cells showed reduced basal respiration, oxygen consumption rate-linked ATP production, maximal and spare capacity, and miR-1 overexpression could prevent impairments in mitochondrial respiration. Our data suggest a role of miR-1 in systemic insulin sensitivity and a new function of miR-1 in regulating mitochondrial respiration in skeletal muscle.

Aims The present investigation evaluated whether pregnancy followed by lactation exerts long-term impacts on maternal hepatic lipid metabolism. Main methods Female mice were subjected to two pregnancies, after which they were either allowed to breastfeed their pups for 21 days (L21) or had their litter removed (L0). Age-matched virgin mice were used as controls (CTL). Three months after the second delivery, serum was collected for lipid profiling, and fragments of liver were used to assess lipid content and to evaluate the key steps of de novo non-esterified fatty acid (NEFA) synthesis, esterification and β-oxidation, very low density lipoprotein (VLDL) assembly and secretion and autophagy. Key findings L0 exhibited a significant increase in hepatic TG and reduced apolipoprotein B-100 (ApoB-100) expression. L21 mice had increased ATP citrate lyase (ACLY) activity and reduced acetyl-CoA carboxylase (ACC) phosphorylation but no increased hepatic TG. On the other hand, L21 mice had reduced hepatic sequestosome 1 (SQSTM1/p62) levels. Increased high density lipoprotein (HDL) cholesterol and hepatic apolipoprotein A-1 (ApoA-1) expression were found exclusively in L21. Significance The present study reveals that long-term hepatic lipid accumulation is induced by the history of pregnancy without lactation. On the other hand, reduced SQSTM1/p62 levels indicate that increased autophagic flux during life may prevent hepatic fat in dams subjected to lactation. Lactation after pregnancy is also obligatory for a long-term increase in maternal HDL. The present data may contribute to the understanding of the mechanisms leading to elevated cardiometabolic risk in women limited to short periods of lactation.

The phenotypes of allergic airway diseases are influenced by the interplay between host genetics and the gut microbiota, which may be modulated by probiotics. We investigated the probiotic effects on allergic inflammation in A/J and C57BL/6 mice. C57BL/6 mice had increased gut microbiota diversity compared to A/J mice at baseline. Acetate producer probiotics differentially modulated and altered the genus abundance of specific bacteria, such as Akkermansia and Allistipes, in mouse strains. We induced airway inflammation followed by probiotic treatment and found that only A/J mice exhibited decreased inflammation, and the beneficial effects of probiotics in A/J mice were partially due to acetate production. To understand the relevance of microbial composition colonization in the development of allergic diseases, we implanted female C57BL/6 mice with A/J embryos to naturally modulate the microbial composition of A/J mice, which increased gut microbiota diversity and reduced eosinophilic inflammation in A/J. These data demonstrate the central importance of microbiota to allergic phenotype severity. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40168-021-01081-2.

Endoplasmic reticulum stress (ERS) and autophagy pathways are implicated in disuse muscle atrophy. The effects of high eicosapentaenoic (EPA) or high docosahexaenoic (DHA) fish oils on soleus muscle ERS and autophagy markers were investigated in a rat hindlimb suspension (HS) atrophy model. Adult Wistar male rats received daily by gavage supplementation (0.3 mL per 100 g b.w.) of mineral oil or high EPA or high DHA fish oils (FOs) for two weeks. Afterward, the rats were subjected to HS and the respective treatments concomitantly for an additional two-week period. After four weeks, we evaluated ERS and autophagy markers in the soleus muscle. Results were analyzed using two-way analysis of variance (ANOVA) and Bonferroni post hoc test. Gastrocnemius muscle ω-6/ω-3 fatty acids (FAs) ratio was decreased by both FOs indicating the tissue incorporation of omega-3 fatty acids. HS altered (p &lt, 0.05) the protein content (decreasing total p38 and BiP and increasing p-JNK2/total JNK2 ratio, and caspase 3) and gene expressions (decreasing BiP and increasing IRE1 and PERK) of ERS and autophagy (decreasing Beclin and increasing LC3 and ATG14) markers in soleus. Both FOs attenuated (p &lt, 0.05) the increase in PERK and ATG14 expressions induced by HS. Thus, both FOs could potentially attenuate ERS and autophagy in skeletal muscles undergoing atrophy.

Background: Sepsis is a life-threatening organ dysfunction associated with unregulated host response to infection. About 20 million people develop sepsis annually, and up to 50% die. Monocytes and macrophages play a key role in the innate and adaptive immune responses but the fully role of these cells in patients with sepsis still remains to be investigated. One of the limitations for the studies of gene expression in monocytes/macrophages in sepsis is the choice of the reference genes. We determined herein the most stable internal gene (s) to investigate gene expressions in monocytes/macrophages of septic patients.Methods: The expression stability of fifteen commonly used reference genes was analyzed by determining the comparative threshold cycle (Ct) values, using the BestKeeper, GeNorm, and NormFinder algorithms.Results: BestKeeper analysis revealed that the syntaxin 5 (STX5A) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) genes are highly stable. GeNorm pointed out STX5A and phosphoglycerate kinase 1 (PGK1) as the most suitable combination, whereas through NormFinder glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 14-3-3 zeta/delta protein (YWHAZ) was the most stable combination. All program analyses discarded the use of heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB). GeNorm and NormFinder indicated actin-beta (ACTB) as the minor stable gene.Conclusions: The combined data indicated that STX5A, PGK1, GAPDH, and HPRT1 are highly suitable reference genes for qPCR analysis of septic patient monocytes. In choosing one reference gene, the results point out STX5A (first place by GeNorm and BestKeeper and third place by NormFinder). This study is the first report on reference genes in freshly obtained monocytes/macrophages from septic patients.

Lutein is an industrially important carotenoid pigment, which is essential for photoprotection and photosynthesis in plants. Lutein is crucial for maintaining human health due to its protective ability from ocular diseases. However, its pathway engineering research has scarcely been performed for microbial production using heterologous hosts, such as Escherichia coli, since the engineering of multiple genes is required. These genes, which include tricky key carotenoid biosynthesis genes typically derived from plants, encode two sorts of cyclases (lycopene ε- and β-cyclase) and cytochrome P450 CYP97C. In this study, upstream genes effective for the increase in carotenoid amounts, such as isopentenyl diphosphate isomerase (IDI) gene, were integrated into the E. coli JM101 (DE3) genome. The most efficient set of the key genes (MpLCYe, MpLCYb and MpCYP97C) was selected from among the corresponding genes derived from various plant (or bacterial) species using E. coli that had accumulated carotenoid substrates. Furthermore, to optimize the production of lutein in E. coli, we introduced several sorts of plasmids that contained some of the multiple genes into the genome-inserted strain and compared lutein productivity. Finally, we achieved 11 mg/l as lutein yield using a mini jar. Here, the high-yield production of lutein was successfully performed using E. coli through approaches of pathway engineering. The findings obtained here should be a base reference for substantial lutein production with microorganisms in the future.

Trypanosoma cruzi, the parasite causing Chagas disease, is a digenetic flagellated protist that infects mammals (including humans) and reduviid insect vectors. Therefore, T. cruzi must colonize different niches in order to complete its life cycle in both hosts. This fact determines the need of adaptations to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. In this regard, it is well known that T. cruzi has a flexible metabolism able to rapidly switch from carbohydrates (mainly glucose) to amino acids (mostly proline) consumption. Also established has been the capability of T. cruzi to use glucose and amino acids to support the differentiation process occurring in the insect, from replicative non-infective epimastigotes to non-replicative infective metacyclic trypomastigotes. However, little is known about the possibilities of using externally available and internally stored fatty acids as resources to survive in nutrient-poor environments, and to sustain metacyclogenesis. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2 production. Additionally, we show that electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid (TCA) cycle, which can be used to feed anabolic pathways such as the de novo biosynthesis of fatty acids. Finally, we show as well that the inhibition of fatty acids mobilization into the mitochondrion diminishes the survival to severe starvation, and impairs metacyclogenesis., Author summary Trypanosoma cruzi is a protist parasite with a life cycle involving two types of hosts, a vertebrate one (which includes humans, causing Chagas disease) and an invertebrate one (kissing bugs, which vectorize the infection among mammals). In both hosts, the parasite faces environmental challenges such as sudden changes in the metabolic composition of the medium in which they develop, severe starvation, osmotic stress and redox imbalance, among others. Because kissing bugs feed infrequently in nature, an intriguing aspect of T. cruzi biology (it exclusively inhabits the digestive tube of these insects) is how they subsist during long periods of starvation. In this work, we show that this parasite performs a metabolic switch from glucose consumption to lipid oxidation, and it is able to consume lipids and the lipid-derived fatty acids from both internal origins as well as externally supplied compounds. When fatty acid oxidation is chemically inhibited by etomoxir, a very well-known drug that inhibits the translocation of fatty acids into the mitochondria, the proliferative insect stage of the parasites has dramatically diminished survival under severe metabolic stress and its differentiation into its infective forms is impaired. Our findings place fatty acids in the centre of the scene regarding their extraordinary resistance to nutrient-depleted environments.

Background Obesity is associated with the development of insulin resistance (IR) and type‐2 diabetes mellitus (T2DM); however, not all patients with T2DM are obese. The Goto–Kakizaki (GK) rat is an experimental model of spontaneous and non‐obese T2DM. There is evidence that the intestine contributes to IR development in GK animals. This information prompted us to investigate small intestine remodeling in this animal model. Methods Four‐month‐old male Wistar (control) and GK rats were utilized for the present study. After removing the small intestine, the duodenum, proximal jejunum, and distal ileum were separated. We then measured villi and muscular and mucosa layer histomorphometry, goblet cells abundance, total myenteric and submucosal neuron populations, and inflammatory marker expression in the small intestinal segments and intestinal transit of both groups of animals. Key Results We found that the GK rats exhibited decreased intestinal area (p, Goto–Kakizaki (GK) rat is an experimental model of spontaneous and non‐obese type‐2 diabetes mellitus—T2DM).GK rats exhibit decreased small intestinal area, increased crypt depth, villi height and thickness, and muscular layer thickness, increased content of IL‐1β and NF‐κB p65, decreased density of submucosal neurons, myenteric and submucosal neuronal and ganglionic hypertrophy, and decreased intestinal transit.The development of IR and T2DM in GK rats is associated with small intestine remodeling with marked changes in morphology, enteric nervous system, and transit.

During its complex life cycle, Trypanosoma cruzi colonizes different niches in its insect and mammalian hosts. This characteristic determined the types of parasites that adapted to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. These T. cruzi stages could be exposed to fatty acids originating from the degradation of the perimicrovillar membrane. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2 production via beta-oxidation. Lastly, we also show that fatty acids are degraded through beta-oxidation. Additionally, through beta-oxidation, electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid cycle, which can be used to feed other anabolic pathways such as the de novo biosynthesis of fatty acids.Author SummaryTrypanosoma cruzi is a protist parasite with a life cycle involving two types of hosts, a vertebrate one (which includes humans, causing Chagas disease) and an invertebrate one (kissing bugs, which vectorize the infection among mammals). In both hosts, the parasite faces environmental challenges such as sudden changes in the metabolic composition of the medium in which they develop, severe starvation, osmotic stress and redox imbalance, among others. Because kissing bugs feed infrequently in nature, an intriguing aspect of T. cruzi biology (it exclusively inhabits the digestive tube of these insects) is how they subsist during long periods of starvation. In this work, we show that this parasite performs a metabolic switch from glucose consumption to lipid oxidation, and it is able to consume lipids and the lipid-derived fatty acids from both internal origins as well as externally supplied compounds. When fatty acid oxidation is chemically inhibited by etomoxir, a very well-known drug that inhibits the translocation of fatty acids into the mitochondria, the proliferative insect stage of the parasites has dramatically diminished survival under severe metabolic stress and its differentiation into its infective forms is impaired. Our findings place fatty acids in the centre of the scene regarding their extraordinary resistance to nutrient-depleted environments.

The progeny of rats born and breastfed by mothers receiving dexamethasone (DEX) during pregnancy exhibits permanent reduction in body weight and adiposity but the precise mechanisms related to this programming are not fully understood. In order to clarify this issue, the present study investigated key aspects of lipoprotein production and lipid metabolism by the liver and the intestine that would explain the reduced adiposity seen in the adult offspring exposed to DEX in utero. Female Wistar rats were treated with DEX (0.1 mg/kg/day) between the 15th and the 21st days of pregnancy, while control mothers were treated with vehicle. Male offspring born to control mothers were nursed by either adoptive control mothers (CTL/CTL) or DEX-treated mothers (CTL/DEX). Male offspring born to DEX-treated mothers were nursed by either control mothers (DEX/CTL) or adoptive DEX-treated mothers (DEX/DEX). We found that only the male DEX/DEX offspring had reduced adiposity. Additionally, male DEX/DEX progeny had lower circulating triacylglycerol (TAG) levels only in fed-state. The four groups of offspring presented similar energy expenditure, respiratory quotient and very low-density lipoprotein (VLDL) production. On the other hand, DEX/DEX rats displayed reduced TAG levels after gavage with olive oil and reduced expression of fatty acid translocase Cd36 (Fat/Cd36) and peroxisome proliferator-activated receptor γ (Pparg) in the jejunum. Altogether, our study supports the notion that reduced fat absorption by the jejunum may contribute to the lower adiposity of the adult offspring born and breastfed by mothers treated with DEX during pregnancy.

This study aimed to investigate the impact of a 16-week dance-based aerobic exercise program on lymphocyte function in healthy and type 2 diabetes mellitus (T2DM) women. We enrolled 23 women: 11 with T2DM and 12 non-diabetic controls. Initially, we performed anthropometry and body composition measurements, afterwards, plasma levels of C-reactive protein, lipids, and glucose were determined. We used flow cytometry to measure the CD25 and CD28 expression in circulating lymphocytes, T-regulatory (Treg) cell percentage, lymphocyte proliferation, and cytokines released by cultured lymphocytes. The T2DM group had a lower proportion of CD28+ cells and a higher percentage of Treg lymphocytes and proliferative capacity at the baseline compared with the control group. After 16 weeks of the program, differences in lymphocytes between the T2DM and the control groups disappeared. The dance program promoted IL-10 increase in both groups. We found decreased IL-4, IL-2, and IL-6 secretion in lymphocytes from the control group and increased IL-17 secretion and IL-10/IL-17 ratio in the T2DM group after the program. The program promoted marked changes in lymphocytes in diabetic women, leading to a balance between the different profiles.

Objective To evaluate the synergic effects of a novel oral supplement formulation, containing prebiotics, yeast β-glucans, minerals and silymarin (Silybum marianum), on lipid and glycidic metabolism, inflammatory and mitochondrial proteins of the liver, in control and high-fat diet-induced obese mice. Methods After an acclimation period, 32 male C57BL/6 mice were divided into the following groups: nonfat diet (NFD) vehicle, NFD supplemented, high-fat diet (HFD) vehicle and HFD supplemented. The vehicle and experimental formulation were administered orally by gavage once a day during the last four weeks of the diet (28 consecutive days). We then evaluated energy homeostasis, inflammation, and mitochondrial protein expression in these groups of mice. Results After four weeks of supplementation, study groups experienced reduced glycemia, dyslipidemia, fat, and hepatic fibrosis levels. Additionally, proliferator-activated receptor-α, AMP-activated protein kinase-1α, peroxisome proliferator-activated receptor γ co-activator -1α, and mitochondrial transcription factor A expression levels were augmented; however, levels of inhibitor of nuclear factor-κB kinase subunit α and p65 nuclear factor-κB expression, and oxidative markers were reduced. Notably, the cortisol/C-reactive protein ratio, a well-characterized marker of the hypothalamic–pituitary–adrenal axis immune interface status, was found to be modulated by the supplement. Conclusion We discovered that the novel supplement was able to modify different antioxidant, metabolic and inflammatory pathways, improving the energy homeostasis and inflammatory status, and consequently alleviated hepatic steatosis.

Fructose consumption by rodents modulates both hepatic and intestinal lipid metabolism and gluconeogenesis. We have previously demonstrated that in utero exposure to dexamethasone (DEX) interacts with fructose consumption during adult life to exacerbate hepatic steatosis in rats. The aim of this study was to clarify if adult rats born to DEX-treated mothers would display differences in intestinal gluconeogenesis after excessive fructose intake. To address this issue, female Wistar rats were treated with DEX during pregnancy and control (CTL) mothers were kept untreated. Adult offspring born to CTL and DEX-treated mothers were assigned to receive either tap water (Control-Standard Chow (CTL-SC) and Dexamethasone-Standard Chow (DEX-SC)) or 10% fructose in the drinking water (CTL-fructose and DEX-fructose). Fructose consumption lasted for 80 days. All rats were subjected to a 40 h fasting before sample collection. We found that DEX-fructose rats have increased glucose and reduced lactate in the portal blood. Jejunum samples of DEX-fructose rats have enhanced phosphoenolpyruvate carboxykinase (PEPCK) expression and activity, higher facilitated glucose transporter member 2 (GLUT2) and facilitated glucose transporter member 5 (GLUT5) content, and increased villous height, crypt depth, and proliferating cell nuclear antigen (PCNA) staining. The current data reveal that rats born to DEX-treated mothers that consume fructose during adult life have increased intestinal gluconeogenesis while recapitulating metabolic and morphological features of the neonatal jejunum phenotype.

Background: Monocytes and macrophages are essential components of the innate and adaptive immune responses and play a critical role in sepsis. Sepsis is a life-threatening organ dysfunction associated with an unregulated host response to infection. About 20 million people develop sepsis annually, and up to 50% die. There is a lack of studies regarding human monocytes and sepsis. This study aimed to determine the most stable internal gene (s) to investigate gene expression in monocytes/macrophages of septic patients.Methods: The expression stability of fifteen commonly used reference genes was analyzed by determining the comparative threshold cycle (Ct) values, using the BestKeeper, GeNorm, and NormFinder algorithms.Results: BestKeeper analysis revealed that the syntaxin 5 (STX5A) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) genes were highly stable. GeNorm pointed out STX5A and phosphoglycerate kinase 1 (PGK1) as the most suitable combination whereas through NormFinder glyceraldehyde 3- phosphate dehydrogenase (GAPDH) and 14-3-3 zeta/delta protein (YWHAZ) was the most stable combination. All programs analysis discarded the use of heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB). GeNorm and NormFinder indicated actin-beta (ACTB) as the least stable gene.Conclusions: The combined data indicated that STX5A, PGK1, GAPDH, and HPRT1 are highly suitable reference genes for qPCR analysis of septic patients monocytes. In the case of choosing one single reference gene, the results point out to STX5A (first place by GeNorm and BestKeeper and third place by NormFinder). This study is the first report on reference genes in monocytes/macrophages from septic patients.

Lifestyle and increased consumption of high fat diets contribute greatly to the development of obesity, insulin resistance, type 2 diabetes (DM2), and cardiovascular diseases, which are less prevalent in young women than in men of the same age or postmenopausal women. One of the consequences of the Western lifestyle and high fat diet is Nonalcoholic Fatty Liver Disease (NAFLD) and its aggressive form, Nonalcoholic Steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular cancer (HCC) and is rapidly becoming the leading cause for end-stage liver disease or liver transplantation. Therefore, we evaluated if female mice were protected against Western Diet (WD)-induced NASH in apolipoprotein E (ApoE) KO animals. Female ApoE KO mice ovariectomized (OVX) or sham operated (SHAM) were fed a WD for 7 weeks. Whole-body fat was increased by ∼65% (P Disclosure G.V. Moreira: None. S.L. Matos: None. F.N. Camargo: None. L. Araujo: None. G.M. Murata: None. C.R.O. Carvalho: None. J. Camporez: None. Funding Fundação de Amparo a Pesquisa do Estado de São Paulo (2018/04956-5)

Unannotated gene sequences in databases are increasing due to sequencing advances. Therefore, computational methods to predict functions of unannotated genes are needed. Moreover, novel enzyme discovery for metabolic engineering applications further encourages annotation of sequences. Here, enzyme functions are predicted using two general approaches, each including several machine learning algorithms. First, Enzyme-models (E-models) predict Enzyme Commission (EC) numbers from amino acid sequence information. Second, Substrate-Enzyme models (SE-models) are built to predict substrates of enzymatic reactions together with EC numbers, and Substrate-Enzyme-Product models (SEP-models) are built to predict substrates, products, and EC numbers. While accuracy of E-models is not optimal, SE-models and SEP-models predict EC numbers and reactions with high accuracy using all tested machine learning-based methods. For example, a single Random Forests-based SEP-model predicts EC first digits with an Average AUC score of over 0.94. Various metrics indicate that the current strategy of combining sequence and chemical structure information is effective at improving enzyme reaction prediction.

Leukocytes are potent regulators of adipose tissue biology and whole-body metabolic homeostasis. In lean, non-obese conditions (insulin-sensitive), adipose tissue has innate and adaptive immune cells, including eosinophils, regulatory T cells, invariant NK cells, and M2 macrophages. A vast expansion in adipose tissue occurs in obesity, and this is associated with a marked alteration in the tissue leukocyte profile. There is a marked increase in B cells, CD8+ T cells, NK cells, neutrophils, and M1 macrophages. This condition induces a state of low-grade, chronic inflammation in the adipose tissue, which disrupts whole-body metabolism. Macrophages were the first leukocyte to be discovered in adipose tissue. Due to their proximity to nearby adipocytes, the macrophages are exposed to high levels of fatty acids and other lipids reported in obesity. Lipid uptake by tissue-resident macrophages is essential for their biological actions. Specifically, lipid uptake and metabolism, particularly of long-chain saturated fatty acids, activate inflammatory signaling pathways, potentiating adipose tissue inflammation, and metabolic dysfunction. Obesity exhibits increased fatty acid levels within the adipose tissue microenvironment. The increased lipid accumulation in the resident macrophages reflects the fatty acid composition of the adipocytes. The dietary fatty acid determines the fatty acid composition of the adipose tissue. Macrophages then accumulate fatty acids indirectly provided by the diet. The composition varies with the acyl chain length, e.g., short-, medium-, or long-chain, and saturated fatty acids. These fatty acids have wide-ranging effects on macrophages. We described herein in detail the impact of the different dietary fatty acids on macrophage functions. Shortly, long-chain saturated fatty acids are pro-inflammatory, whereas medium-chain fatty acids are relatively benign. Long-chain unsaturated fatty acids often antagonize the pro-inflammatory effects of long-chain saturated fatty acids.

Background/Aims: Obesity is a risk factor associated with cardiometabolic complications. Recently, we reported that miRNA-22 deletion attenuated high-fat diet-induced adiposity and prevented dyslipidemia without affecting cardiac hypertrophy in male mice. In this study, we examined the impact of miRNA-22 in obesogenic diet-induced cardiovascular and metabolic disorders in females. Methods: Wild type (WT) and miRNA-22 knockout (miRNA-22 KO) females were fed a control or an obesogenic diet. Body weight gain, adiposity, glucose tolerance, insulin tolerance, and plasma levels of total cholesterol and triglycerides were measured. Cardiac and white adipose tissue remodeling was assessed by histological analyses. Echocardiography was used to evaluate cardiac function and morphology. RNA-sequencing analysis was employed to characterize mRNA expression profiles in female hearts. Results: Loss of miRNA-22 attenuated body weight gain, adiposity, and prevented obesogenic diet-induced insulin resistance and dyslipidemia in females. WT obese females developed cardiac hypertrophy. Interestingly, miRNA-22 KO females displayed cardiac hypertrophy without left ventricular dysfunction and myocardial fibrosis. Both miRNA-22 deletion and obesogenic diet changed mRNA expression profiles in female hearts. Enrichment analysis revealed that genes associated with regulation of the force of heart contraction, protein folding and fatty acid oxidation were enriched in hearts of WT obese females. In addition, genes related to thyroid hormone responses, heart growth and PI3K signaling were enriched in hearts of miRNA-22 KO females. Interestingly, miRNA-22 KO obese females exhibited reduced mRNA levels of Yap1, Egfr and Tgfbr1 compared to their respective controls. Conclusion: This study reveals that miRNA-22 deletion induces cardiac hypertrophy in females without affecting myocardial function. In addition, our findings suggest miRNA-22 as a potential therapeutic target to treat obesity-related metabolic disorders in females.

Agomelatine (AGO) is an antidepressant drug with agonistic activity at melatonin receptor 1 (MT1) and MT2 and with neutral antagonistic activity at serotonin receptor 5-HT2C. Although experimental studies show that melatonin reduces hypertriglyceridemia and hepatic steatosis induced by excessive fructose intake, no studies have tested if AGO exerts similar actions. To address this issue we have treated male Wistar rats with fructose (15% in the drinking water) and/or AGO (40 mg/kg/day) for two weeks. AGO reduced body weight gain, feeding efficiency and hepatic lipid levels without affecting caloric intake in fructose-treated rats. AGO has also decreased very low-density lipoprotein (VLDL) production and circulating TAG levels after an oral load with olive oil. Accordingly, treatment with AGO reduced the hepatic expression of fatty acid synthase (Fasn), a limiting step for hepatic de novo lipogenesis (DNLG). The expression of apolipoprotein B (Apob) and microsomal triglyceride transfer protein (Mttp) in the ileum, two crucial proteins for intestinal lipoprotein production, were also downregulated by treatment with AGO. Altogether, the present data show that AGO mimics the metabolic benefits of melatonin when used in fructose-treated rats. This study also suggests that it is relevant to evaluate the potential of AGO to treat metabolic disorders in future clinical trials.

Background Skeletal muscle changes contribute to reduced physical performance after myocardial infarction (MI). Exercise has been recommended to stable patients. However, the effects of resistance exercise after MI are not clear. We compared the effects of aerobic and resistance exercise initiated during compensated cardiac remodeling in infarcted rat gastrocnemius muscle. Methods Three months after MI induction, Wistar rats were divided into four groups: Sham (n=20); sedentary MI (MI-S, n=9); aerobic exercised MI (MI-A, n=9); and resistance exercised MI (MI-R, n=13). Exercised rats trained three times a week for 12 weeks on a treadmill or ladder. Energy metabolism, oxidative stress markers, and antioxidant enzyme activities were assessed by spectrophotometry. Satellite cells activation markers (MyoD, NCAM, and myosin heavy chain neonatal isoform) were assessed by immunofluorescence or Western blot (Pax-7). Statistical analysis: ANOVA or Mann Whitney. Results Physical aerobic capacity was greater in MI-A and strength gain higher in MI-R. Cardiac structures and left ventricular function evaluated by echocardiogram did not differ between infarcted groups. Histological analysis showed that MI size and gastrocnemius cross sectional area did not differ between infarcted groups. Oxygen reactive species production was higher in MI-S than Sham and lipid hydroperoxide concentration was lower in MI-A than the other groups. Catalase activity was higher and glutathione peroxidase lower in infarcted groups than Sham. Superoxide dismutase activity was higher in Sham and MI-R than MI-S. Skeletal muscle metabolism enzyme activity did not differ between groups, except for increase pyruvate kinase in MI-S against the other groups, and β-hydroxyacyl CoA dehydrogenase in MI-S against Sham. Satellite cell activation and protein expression of MAPK and NF-kB did not differ between groups. Conclusion Aerobic and resistance exercise respectively improves physical capacity and muscle strength without changing echocardiographic parameters of infarcted rats. Myocardial infarction increases oxygen reactive species production and changes antioxidant enzyme activity and glucose and fatty acid metabolism. Aerobic exercise is superior to resistance exercise against oxidative stress reducing muscle lipid hydroperoxide concentration and attenuating change in glutathione peroxidase activity. Acknowledgement/Funding Financial support: Fapesp, CNPq, Capes, and UNESP

Several pathological conditions predict the use of glucocorticoids for the management of the inflammatory response; however, chronic or high dose glucocorticoid treatment is associated with hyperglycemia, hyperlipidemia, and insulin resistance and can be considered a risk factor for cardiovascular disease. Therefore, we investigated the mechanisms involved in the vascular responsiveness and inflammatory profile of mesenteric arteries of rats treated with high doses of glucocorticoids. Wistar rats were divided into a control (CO) group and a dexamethasone (DEX) group, that received dexamethasone for 7 days (2mg/kg/day, i.p.). Blood samples were used to assess the lipid profile and insulin tolerance. Vascular reactivity to Phenylephrine (Phe) and insulin, and O2•-production were evaluated. The intracellular insulin signaling pathway PI3K/AKT/eNOS and MAPK/ET-1 were investigated. Regarding the vascular inflammatory profile, TNF-α, IL-6, IL-1β and IL-18 were assessed. Dexamethasone-treated rats had decreased insulin tolerance test and endothelium-dependent vasodilation induced by insulin. eNOS inhibition caused vasoconstriction in the DEX group, which was abolished by the ET-A antagonist. Insulin-mediated relaxation in the DEX group was restored in the presence of the O2.- scavenger TIRON. Nevertheless, in the DEX group there was an increase in Phe-induced vasoconstriction. In addition, the intracellular insulin signaling pathway PI3K/AKT/eNOS was impaired, decreasing NO bioavailability. Regarding superoxide anion generation, there was an increase in the DEX group, and all measured proinflammatory cytokines were also augmented in the DEX group. In addition, the DEX-group presented an increase in low-density lipoprotein cholesterol (LDL-c) and total cholesterol (TC) and reduced high-density lipoprotein cholesterol (HDL-c) levels. In summary, treatment with high doses of dexamethasone promoted changes in insulin-induced vasodilation, through the reduction of NO bioavailability and an increase in vasoconstriction via ET-1 associated with generation of O2•- and proinflammatory cytokines.

Pioglitazone belongs to the class of drugs thiazolidinediones (TZDs) and is an oral hypoglycemic drug, used in the treatment of type 2 diabetes, which improves insulin sensitivity in target tissues. Adipose tissue is the main target of pioglitazone, a PPARg and PPARa agonist; however, studies also point to skeletal muscle as a target. Non-PPAR targets of TZDs have been described, thus we aimed to study the direct effects of pioglitazone on skeletal muscle and the possible role of microRNAs as targets of this drug. Pioglitazone treatment of obese mice increased insulin-mediated glucose transport as a result of increased fatty acid oxidation and mitochondrial activity. PPARg blockage by treatment with GW9662 nullified pioglitazone's effect on systemic and muscle insulin sensitivity and citrate synthase activity of obese mice. After eight weeks of high-fat diet, miR-221–3p expression in soleus muscle was similar among the groups and miR-23b-3p and miR-222–3p were up-regulated in obese mice compared to the control group, and treatment with pioglitazone was able to reverse this condition. In vitro studies in C2C12 cells suggest that inhibition of miR-222–3p protects C2C12 cells from insulin resistance and increased non-mitochondrial respiration induced by palmitate. Together, these data demonstrate a role of pioglitazone in the downregulation of microRNAs that is not dependent on PPARg. Moreover, miR-222 may be a novel PPARg-independent mechanism through which pioglitazone improves insulin sensitivity in skeletal muscle.

Background Obesity can lead to a chronic systemic inflammatory state that increases the risk of cancer development. Therefore, this study aimed to evaluate the alterations in tumor non-infiltrated lymphocytes function and melanoma growth in animals maintained on a high-fat diet and/or moderate physical exercise program in a murine model of melanoma. Methods Female mice were randomly divided into eight groups: 1) normolipidic control (N), 2) normolipidic + melanoma (NM), 3) high-fat control (H), 4) high-fat + melanoma (HM), 5) normolipidic control + physical exercise (NE), 6) normolipidic melanoma + physical exercise (NEM), 7) high-fat control + physical exercise (HE), and 8) high-fat melanoma + physical exercise (HEM). After 8 weeks of diet treatment and/or moderate physical exercise protocol, melanoma was initiated by explanting B16F10 cells into one-half of the animals. Results Animals fed a high-fat diet presented high-energy consumption (30%) and body weight gain (H and HE vs N and NE, 37%; HM and HEM vs NM and NEM, 73%, respectively), whether or not they carried melanoma explants. Although the tumor growth rate was higher in animals from the HM group than in animals from any other sedentary group, it was reduced by the addition of a physical exercise regimen. We also observed an increase in stimulated peripheral lymphocyte proliferation and a decrease in the T-helper 1 response in the HEM group. Conclusions The results of the present study support the hypothesis that altering function of tumor non-infiltrated lymphocytes via exercise-related mechanisms can slow melanoma progression, indicating that the incorporation of a regular practice of moderate-intensity exercises can be a potential strategy for current therapeutic regimens in treating advanced melanoma.

The effect of a short-term creatine supplementation on hindlimb suspension (HS)-induced muscle atrophy was investigated. Creatine monohydrate (5 g/kg b.w. per day) or placebo, divided in 2 daily doses, was given by oral gavage for 5 days. Rats were maintained in HS with dietary supplementation concomitantly for 5 days. Body weight, soleus and EDL muscle masses, and cross-sectional areas (CSA) of the muscle fibers were measured. Signaling pathways associated with skeletal muscle mass regulation (FST, MSTN, FAK, IGF-1, MGF, Akt, mTOR, atrogin-1, and MuRF1 expressions, and Akt, S6, GSK3B, and 4EBP1 proteins) were evaluated in the muscles. Soleus muscle exhibited more atrophy than the EDL muscle due to HS. Creatine supplementation attenuated the decrease of wet weight and increased p-4EBP1 protein in the EDL muscle of HS rats. Also, creatine increased mTOR and atrogin-1 expressions in the same muscle and condition. In the absence of HS, creatine supplementation increased FAK and decreased MGF expressions in the EDL muscle. Creatine attenuated the increase in FST expression due to HS in the soleus muscle. MuRF1 expression increased in the soleus muscle due to creatine supplementation in HS animals whereas atrogin-1 expression increased still further in this group compared with untreated HS rats. In conclusion, short-term creatine supplementation changed protein metabolism signaling in soleus and EDL muscles. However, creatine supplementation only slightly attenuated the mass loss of both muscles and did not prevent the CSA reduction and muscle strength decrease induced by HS for 5 days.

Background/aims Skeletal mass loss is reported in several catabolic conditions and it has been associated with a reduced intracellular L-glutamine content. We investigated the association of intracellular L-glutamine concentration with the protein content in skeletal muscle cells. Methods We cultivated C2C12 myotubes in the absence or presence of 2 (reference condition), 8 or 16 mM L-glutamine for 48 hours, and the variations in the contents of amino acids and proteins measured. We used an inhibitor of L-glutamine synthesis (L-methionine sulfoximine - MSO) to promote a further reduction in intracellular L-glutamine levels. Amino acids contents in cells and media were measured using LC-MS/MS. We measured changes in phosphorylated Akt, RP-S6, and 4E-BP1contents in the absence or presence of insulin by western blotting. Results Reduced intracellular L-glutamine concentration was associated with decreased protein content and increased protein breakdown. Low intracellular glutamine levels were also associated with decreased p-Akt contents in the presence of insulin. A further decrease in intracellular L-glutamine caused by glutamine synthetase inhibitor reduced protein content and levels of amino acids generated from glutamine metabolism and increased bAib still further. Cells exposed to high medium glutamine levels did not have any change in protein content but exhibited increased contents of the amino acids derived from L-glutamine metabolism. Conclusion Intracellular L-glutamine levels per se play a role in the control of protein content in skeletal muscle myotubes.

L-Glutamine (L-Gln) supplementation has been pointed out as an anticatabolic intervention, but its effects on protein synthesis and degradation signaling in skeketal muscle are still poorly known. The effects of L-Gln pretreatment (1 g kg-1 day-1 body weight for 10 days) on muscle fiber cross-sectional area (CSA), amino acid composition (measured by LC-MS/MS) and protein synthesis (Akt-mTOR) and degradation (ubiquitin ligases) signaling in soleus and extensor digitorum longus (EDL) muscles in 24-h-fasted mice were investigated. The fiber CSA of EDL muscle was not different between the L-Gln-fasted and L-Gln-fed groups. This finding was associated with reduced contents of L-Leu and L-Iso and activation of protein synthesis signaling (p-RPS6Ser240/244 and Akt-mTOR). The spectrum of soleus muscle fiber CSA distribution was larger in L-Gln-fasted as compared with placebo-fasted mice. This effect of L-Gln pretreatment was associated with changes in red fibers L-Gln metabolism as indicated by increased intracellular L-glutamine/L-glutamate ratio, L-aspartate and GABA levels. L-Gln supplementation reduced fasting-induced mass loss in tibialis anterior and gastrocnemius muscles. Evidence is presented that pretreatment with L-glutamine attenuates skeletal muscle atrophy induced by 24-h fasting through mechanisms that vary with the muscle fiber type.

Distinct environmental insults might interact with fructose consumption and contribute to the development of metabolic disorders. To address whether in utero glucocorticoid exposure and fructose intake modulate metabolic responses, adult female Wistar rats were exposed to dexamethasone (DEX) during pregnancy, and the offspring were administered fructose at a later time. Briefly, dams received DEX during the third period of pregnancy, while control dams remained untreated. Offspring born to control and DEX-treated mothers were defined as CTL-off and DEX-off, respectively, while untreated animals were designated CTL-off-CTL and DEX-off-CTL. CLT-off and DEX-off treated with 10% fructose in the drinking water for 8 weeks are referred to as CTL-off-FRU and DEX-off-FRU. We found that fructose promoted glucose intolerance and whole-body gluconeogenesis in both CTL-off-FRU and DEX-off-FRU animals. On the other hand, hepatic lipid accumulation was significantly stimulated in DEX-off-FRU rats when compared to the CTL-off-FRU group. The DEX-off-FRU group also displayed impaired very-low-density lipoprotein (VLDL) production and reduced hepatic expression of apoB, mttp, and sec22b. DEX-off-FRU has lower hepatic levels of autophagy markers. Taken together, our results support the unprecedented notion that in utero glucocorticoid exposure exacerbates hepatic steatosis caused by fructose consumption later in life.