Metabolic features of neutrophilic differentiation of HL-60 cells in hyperglycemic environments.

INTRODUCTION: Chronic hyperglycemia affects neutrophil functions, leading to reduced pathogen killing and increased morbidity. This impairment has been directly linked to increased glycemia, however, how this specifically affects neutrophils metabolism and their differentiation in the bone marrow is unclear and difficult to study. RESEARCH DESIGN AND METHODS: We used high-resolution respirometry to investigate the metabolism of resting and activated donor neutrophils, and flow cytometry to measure surface CD15 and CD11b expression. We then used HL-60 cells differentiated towards neutrophil-like cells in standard media and investigated the effect of doubling glucose concentration on differentiation metabolism. We measured the oxygen consumption rate (OCR), and the enzymatic activity of carnitine palmitoyl transferase 1 (CPT1) and citrate synthase during neutrophil-like differentiation. We compared the surface phenotype, functions, and OCR of neutrophil-like cells differentiated under both glucose concentrations. RESULTS: Donor neutrophils showed significant instability of CD11b and OCR after phorbol 12-myristate 13-acetate stimulation at 3 hours post-enrichment. During HL-60 neutrophil-like cell differentiation, there was a significant increase in surface CD15 and CD11b expression together with the loss of mitochondrial mass. Differentiated neutrophil-like cells also exhibited higher CD11b expression and were significantly more phagocytic. In higher glucose media, we measured a decrease in citrate synthase and CPT1 activities during neutrophil-like differentiation. CONCLUSIONS: HL-60 neutrophil-like differentiation recapitulated known molecular and metabolic features of human neutrophil differentiation. Increased glucose concentrations correlated with features described in hyperglycemic donor neutrophils including increased CD11b and phagocytosis. We used this model to describe metabolic features of neutrophil-like cell differentiation in hyperglycemia and show for the first time the downregulation of CPT1 and citrate synthase activity, independently of mitochondrial mass.

The implications of exercise in Drosophila melanogaster: insights into Akt/p38 MAPK/Nrf2 pathway associated with Hsp70 regulation in redox balance maintenance.

This study investigated the potential effects of exercise on the responses of energy metabolism, redox balance maintenance, and apoptosis regulation in Drosophila melanogaster to shed more light on the mechanisms underlying the increased performance that this emerging exercise model provides. Three groups were evaluated for seven days: the control (no exercise or locomotor limitations), movement-limited flies (MLF) (no exercise, with locomotor limitations), and EXE (with exercise, no locomotor limitations). The EXE flies demonstrated greater endurance-like tolerance in the swimming test, associated with increased citrate synthase activity, lactate dehydrogenase activity and lactate levels, and metabolic markers in exercise. Notably, the EXE protocol regulated the Akt/p38 MAPK/Nrf2 pathway, which was associated with decreased Hsp70 activation, culminating in glutathione turnover regulation. Moreover, reducing the locomotion environment in the MLF group decreased endurance-like tolerance and did not alter citrate synthase activity, lactate dehydrogenase activity, or lactate levels. The MLF treatment promoted a pro-oxidant effect, altering the Akt/p38 MAPK/Nrf2 pathway and increasing Hsp70 levels, leading to a poorly-regulated glutathione system. Lastly, we demonstrated that exercise could modulate major metabolic responses in Drosophila melanogaster aerobic and anaerobic metabolism, associated with apoptosis and cellular redox balance maintenance in an emergent exercise model.

Kombucha tea improves glucose tolerance and reduces hepatic steatosis in obese mice.

Nonalcoholic fatty liver disease (NAFLD), often associated with obesity, is becoming one of the most common liver diseases worldwide. It is estimated to affect one billion individuals and may be present in approximately 25% of the population globally. NAFLD is viewed as a hepatic manifestation of metabolic syndrome, with humans and animal models presenting dyslipidemia, hypertension, and diabetes. The gut-liver axis has been considered the main pathogenesis branch for NAFLD development. Considering that foods or beverages could modulate the gastrointestinal tract, immune system, energy homeostasis regulation, and even the gut-liver axis, we conducted an exploratory study to analyze the effects of kombucha probiotic on hepatic steatosis, glucose tolerance, and hepatic enzymes involved in carbohydrate and fat metabolism using a pre-clinical model. The diet-induced obese mice presented glucose intolerance, hyperinsulinemia, hepatic steatosis, increased collagen fiber deposition in liver vascular spaces, and upregulated TNF-alpha and SREBP-1 gene expression. Mice receiving the kombucha supplement displayed improved glucose tolerance, reduced hyperinsulinemia, decreased citrate synthase and phosphofructokinase-1 enzyme activities, downregulated G-protein-coupled bile acid receptor, also known as TGR5, and farnesol X receptor gene expression, and attenuated steatosis and hepatic collagen fiber deposition. The improvement in glucose tolerance was accompanied by the recovery of acute insulin-induced liver AKT serine phosphorylation. Thus, it is possible to conclude that this probiotic drink has a beneficial effect in reducing the metabolic alterations associated with diet-induced obesity. This probiotic beverage deserves an extension of studies to confirm or refute its potentially beneficial effects.

The Taurine-Conjugated Bile Acid (TUDCA) Normalizes Insulin Secretion in Pancreatic ß-Cells Exposed to Fatty Acids: The Role of Mitochondrial Metabolism.

Bile acid tauroursodeoxycholic (TUDCA), formed from the association of ursodeoxycholic acid (UDCA) with taurine, has already been shown to increase mitochondrial biogenesis and cell survival, in addition to reduce reticulum stress markers in different cell types. However, its mechanism of action upon insulin secretion control in obesity is still unknown. In this sense, we seek to clarify whether taurine, associated with bile acid, could improve the function of the pancreatic ß-cells exposed to fatty acids through the regulation of mitochondrial metabolism. To test this idea, insulin-producing cells (INS1-E) were exposed to a fatty acid mix containing 500 µM of each palmitate and oleate for 48 hours treated or not with 300 µM of TUDCA. After that, glucose-stimulated insulin secretion and markers of mitochondrial metabolism were evaluated. Our results showed that the fatty acid mix was efficient in inducing hyperfunction of INS1-E cells as observed by the increase in insulin secretion, protein expression of citrate synthase, and mitochondrial density, without altering cell viability. The treatment with TUDCA normalized insulin secretion, reducing the protein expression of citrate synthase, mitochondrial mass, and the mitochondrial membrane potential. This effect was associated with a decrease in the generation of mitochondrial superoxide and c-Jun N-terminal kinase (JNK) protein content. The findings are also consistent with the hypothesis that TUDCA normalizes insulin secretion by improving mitochondrial metabolism and redox balance. Thus, it highlights likely mechanisms of the action of this bile acid on the glycemic homeostasis reestablishment in obesity.

Physical exercise improves mitochondrial function in ovariectomized rats.

Estrogen deficiency causes metabolic disorders in humans and rodents, including in part due to changes in energy expenditure. We have shown previously that skeletal muscle mitochondrial function is compromised in ovariectomized (Ovx) rats. Since physical exercise is a powerful strategy to improve skeletal muscle mitochondrial content and function, we hypothesize that exercise training would counteract the deficiency-induced skeletal muscle mitochondrial dysfunction in Ovx rats. We report that exercised Ovx rats, at 60-65% of maximal exercise capacity for 8 weeks, exhibited less fat accumulation and body weight gain compared with sedentary controls. Treadmill exercise training decreased muscle lactate production, indicating a shift to mitochondrial oxidative metabolism. Furthermore, reduced soleus muscle mitochondrial oxygen consumption confirmed that estrogen deficiency is detrimental to mitochondrial function. However, exercise restored mitochondrial oxygen consumption in Ovx rats, achieving similar levels as in exercised control rats. Exercise-induced skeletal muscle peroxisome proliferator-activated receptor-γ coactivator-1α expression was similar in both groups. Therefore, the mechanisms by which exercise improves mitochondrial oxygen consumption appears to be different in Ovx-exercised and sham-exercised rats. While there was an increase in mitochondrial content in sham-exercised rats, demonstrated by a greater citrate synthase activity, no induction was observed in Ovx-exercised rats. Normalizing mitochondrial respiratory capacity by citrate synthase activity indicates a better oxidative phosphorylation efficiency in the Ovx-exercised group. In conclusion, physical exercise sustains mitochondrial function in ovarian hormone-deficient rats through a non-conventional mitochondrial content-independent manner.

Lactate modulates cardiac gene expression in mice during acute physical exercise.

The aim of the present study was to verify the role of lactate as a signaling molecule in cardiac tissue under physiological conditions. C57BL6/J male mice were submitted to acute running bouts on a treadmill at different exercise intensities (30, 60, and 90% of maximal speed - Smax) under the effect of two doses (0.5 and 5 mM) of α-cyano-4-hydroxycynnamate (CINN), a blocker of lactate transporters. Cardiac lactate levels, activity of the enzymes of glycolytic [hexokinase (HK) and lactate dehydrogenase (LDH)] and oxidative metabolism [citrate synthase (CS)], and expression of genes also related to metabolism [LDH, nuclear factor erythroid 2-related factor 2 (NRF-2), cytochrome oxidase IV (COX-IV), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)] were evaluated. Elevated cardiac lactate levels were observed after high intensity running at 90% of Smax, which were parallel to increased activity of the HK and CS enzymes and mRNA levels of PGC-1α and COX-IV. No changes were observed in cardiac lactate levels in mice running at lower exercise intensities. Interestingly, prior intraperitoneal administration (15 min) of CINN (0.5 mM) significantly reduced cardiac lactate concentration, activities of HK and CS, and mRNA levels of PGC-1α and COX-IV in mice that ran at 90% of Smax. In addition, cardiac lactate levels were significantly correlated to both PGC-1α and COX-IV cardiac gene expression. The present study provides evidence that cardiac lactate levels are associated to gene transcription during an acute bout of high intensity running exercise.

Aerobic Exercise Training Prevents Insulin Resistance and Hepatic Lipid Accumulation in LDL Receptor Knockout Mice Chronically Fed a Low-Sodium Diet.

BACKGROUND: A low-sodium (LS) diet reduces blood pressure, contributing to the prevention of cardiovascular diseases. However, intense dietary sodium restriction impairs insulin sensitivity and worsens lipid profile. Considering the benefits of aerobic exercise training (AET), the effect of LS diet and AET in hepatic lipid content and gene expression was investigated in LDL receptor knockout (LDLr-KO) mice. METHODS: Twelve-week-old male LDLr-KO mice fed a normal sodium (NS) or LS diet were kept sedentary (S) or trained (T) for 90 days. Body mass, plasma lipids, insulin tolerance testing, hepatic triglyceride (TG) content, gene expression, and citrate synthase (CS) activity were determined. Results were compared by 2-way ANOVA and Tukey's post-test. RESULTS: Compared to NS, LS increased body mass and plasma TG, and impaired insulin sensitivity, which was prevented by AET. The LS-S group, but not the LS-T group, presented greater hepatic TG than the NS-S group. The LS diet increased the expression of genes related to insulin resistance (ApocIII, G6pc, Pck1) and reduced those involved in oxidative capacity (Prkaa1, Prkaa2, Ppara, Lipe) and lipoprotein assembly (Mttp). CONCLUSION: AET prevented the LS-diet-induced TG accumulation in the liver by improving insulin sensitivity and the expression of insulin-regulated genes and oxidative capacity.

Caenorhabditis elegans as a model for studies on quinolinic acid-induced NMDAR-dependent glutamatergic disorders.

Quinolinic acid (QUIN) is an agonist of the neurotransmitter glutamate (Glu) capable of binding to N-methyl-D-aspartate receptors (NMDAR) increasing glutamatergic signaling. QUIN is known for being an endogenous neurotoxin, able to induce neurodegeneration. In Caenorhabditis elegans, the mechanism by which QUIN induces behavioral and metabolic toxicity has not been fully elucidated. The effects of QUIN on behavioral and metabolic parameters in nmr-1 and nmr-2 NMDA receptors in transgenic and wild-type (WT) worms were performed to decipher the pathway by which QUIN exerts its toxicity. QUIN increased locomotion parameters such as wavelength and movement amplitude medium, as well as speed and displacement, without modifying the number of body bends in an NMDAR-dependent-manner. QUIN increased the response time to the chemical stimulant 1-octanol, which is modulated by glutamatergic neurotransmission in the ASH neuron. Brood size increased after exposure to QUIN, dependent upon nmr-2/NMDA-receptor, with no change in lifespan. Oxygen consumption, mitochondrial membrane potential, and the flow of coupled and unbound electrons to ATP production were reduced by QUIN in wild-type animals, but did not alter citrate synthase activity, altering the functionality but the mitochondrial viability. Notably, QUIN modified fine locomotor and chemosensory behavioral parameters, as well as metabolic parameters, analogous to previously reported effects in mammals. Our results indicate that QUIN can be used as a neurotoxin to elicit glutamatergic dysfunction in C. elegans in a way analogous to other animal models.

Load-matched acute and chronic exercise induce changes in mitochondrial biogenesis and metabolic markers.

We investigated the effects of acute and chronic exercise, prescribed in different intensity zones, but with total load-matched on mitochondrial markers (cytochrome C oxidase subunit IV (COX-IV), mitochondrial transcription factor A (Tfam), and citrate synthase (CS) activity in skeletal muscles, heart, and liver), glycogen stores, aerobic capacity, and anaerobic index in swimming rats. For this, 2 experimental designs were performed (acute and chronic efforts). Load-matched exercises were prescribed below, above, and on the anaerobic threshold (AnT), determined by the lactate minimum test. In chronic programs, 2 training prescription strategies were assessed (monotonous and linear periodized model). Results show changes in glycogen stores but no modification in the COX-IV and Tfam contents after acute exercises. In the chronic protocols, COX-IV and Tfam proteins and CS adaptations were intensity- and tissue-dependent. Monotonous training promoted better adaptations than the periodized model. Training at 80% of the AnT improved both performance variables, emphasizing the anaerobic index, concomitant to CS and COX-IV improvement (soleus muscle). The aerobic capacity and CS activity (gastrocnemius) were increased after 120% AnT training. In conclusion, acute exercise protocol did not promote responses in mitochondrial target proteins. An intensity and tissue dependence were reported in the chronic protocols, highlighting training at 80 and 120% of the AnT. Novelty: Load-matched acute exercise did not enhance COX-IV and Tfam contents in skeletal muscles, heart, and liver. In chronic exercise, COX-IV, Tfam, and CS activity adaptations were intensity- and tissue-dependent. Monotonous training was more efficient than the periodized linear model in adaptations of target proteins and enzymatic activity.

Swimming Program on Mildly Diabetic Rats in Pregnancy.

The present study aims to confirm if the moderate-intensity swimming has successful glycemic control and non-toxic oxidative stress levels and to verify the influence on pancreatic adaptations, embryo implantation, and placental efficiency. Female Wistar rats were randomly distributed to obtain mildly diabetic by streptozotocin induction at birth and the non-diabetic females given vehicle. At adulthood, pregnant rats were put at random into sedentary non-diabetic rats (ND); exercise non-diabetic rats (NDEx); sedentary diabetic rats (D); and exercise diabetic rats (DEx). The rats of the groups submitted to moderate intensity carried loads equivalent to 4% of body weight. On day 17 of gestational day, all rats were submitted to oral glucose tolerance test (OGTT). Next day (GD18), the rats were anesthetized and killed to count implantation sites and to collect placentas, blood, and muscle samples for biochemical biomarkers and pancreas for immunohistochemical analysis. The moderate exercise used was not sufficient to stimulate the aerobic pathway but presented positive results on glucose metabolism, lower embryo postimplantation loss, and pancreatic morphology compared with the sedentary diabetic group. However, the DEx group showed muscular damage, decreased antioxidant defense, and lipid peroxidation. Thus, the moderate-intensity exercise reduces glycemic levels during OGTT and causes no damage to non-diabetic rats related to other analyzed parameters in this study. The exercised diabetic rats present better glycemic metabolism in OGTT, islet pancreatic morphology, and embryofetal development. However, it is necessary an adjustment in this exercise intensity to improve the effectiveness of aerobic training for reduction of maternal muscular and lipid membrane damages.

Metabolic status of the coral Mussismilia harttii in field conditions and the effects of copper exposure in vitro.

It is widely known that metals can alter enzyme functioning, however, little is known about the mechanisms of metal toxicity in energy metabolism enzymes of corals. Thus, the present study had two objectives: firstly, we evaluated the activity of eight metabolic enzymes of the coral Mussismilia harttii to clarify metabolic functioning under field conditions. After that, we investigated the in vitro effect of copper (Cu) exposure in the activity of an enzyme representative of each metabolism stage. We evaluated enzymes involved in glycolysis (hexokinase, HK; phosphofructokinase, PFK; pyruvate kinase, PK and lactate dehydrogenase, LDH), Krebs cycle (citrate synthase, CS and isocitrate dehydrogenase, IDH), electron transport chain (electron transport system activity, ETS) and pentose phosphate pathway (glucose-6-phosphate dehydrogenase, G6PDH). The in vitro tests were performed through contamination of the reaction medium using Cu concentrations of 0, 1.4, 3.7 and 14.2 µg L-1. The results showed that M. harttii has elevated activity of HK, PK and CS in field conditions compared to the activity of other energy metabolism enzymes evaluated. Moreover, lower activities of LDH and ETS in exposed samples were observed. In conclusion, in field conditions this species has elevated aerobic metabolism and glucose may be an important energetic fuel. Also, exposure to Cu in vitro caused inhibition of LDH and ETS by direct binding.

Early life fluoxetine treatment causes long-term lean phenotype in skeletal muscle of rats exposed to maternal lard-based high-fat diet.

There is a concern about early life exposure to Selective Serotonin Reuptake Inhibitors (SSRI) in child development and motor system maturation. Little is known, however, about the interaction of environmental factors, such as maternal nutrition, associated with early exposure to SSRI. The increased maternal consumption of high-fat diets is worrisome and affects serotonin system development with repercussions in body phenotype. This study aimed to assess the short- and long-term effects of neonatal fluoxetine treatment on the body and skeletal muscle phenotype of rats exposed to a maternal lard-based high-fat (H) diet during the perinatal period. A maternal lard-based high-fat diet causes reduced birth weight, a short-term reduction in type IIA fibers in the soleus muscle, and in type IIB fibers in the Extensor Digitorum Longus (EDL) muscle, reducing Lactate Dehydrogenase (LDH) activity in both muscles. In the long-term, the soleus showed reduced muscle weight, smaller area and perimeter of muscle fibers, while the EDL muscle showed reduced Citrate Synthase (CS) activity in offspring from the rats on the maternal lard-based high-fat diet. Early-life exposure to fluoxetine reduced body weight and growth and reduced soleus weight and enzymatic activity in young rats. Exposure to neonatal fluoxetine in adult rats caused a decreased body mass index, less food intake, and reduced muscle weight with reduced CS and LDH activity. Neonatal fluoxetine in young rats exposed to a maternal lard-based high-fat diet caused reduced body weight and growth, reduced soleus weight as well as area and perimeter of type I muscle fibers. In adulthood, there was a reduction in food intake, increased proportion of IIA type fibers, reduced area and perimeter of type IIB, and reduction in levels of CS activity in EDL muscle. Neonatal fluoxetine treatment in rats exposed to a maternal lard-based, high-fat diet induces a reduction in muscle weight, an increase in the proportion of oxidative fibers and greater oxidative enzymatic activity in adulthood.

A single session of physical activity restores the mitochondrial organization disrupted by obesity in skeletal muscle fibers.

BACKGROUND: Several studies have proved that physical activity (PA) regulates energetic metabolism associated with mitochondrial dynamics through AMPK activation in healthy subjects. Obesity, a condition that induces oxidative stress, mitochondrial dysfunction, and low AMPK activity leads to mitochondrial fragmentation. However, few studies describe the effect of PA on mitochondrial dynamics regulation in obesity. AIM: The present study aimed to evaluate the effect of a single session of PA on mitochondrial dynamics regulation as well as its effect on mitochondrial function and organization in skeletal muscles of obese rats (Zucker fa/fa). MAIN METHODS: Male Zucker lean and Zucker fa/fa rats aged 12 to 13 weeks were divided into sedentary and subjected-to-PA (single session swimming) groups. Gastrocnemius muscle was dissected into isolated fibers, mitochondria, mRNA, and total proteins for their evaluation. KEY FINDINGS: The results showed that PA increased the Mfn-2 protein level in the lean and obese groups, whereas Drp1 levels decreased in the obese group. OMA1 protease levels increased in the lean group and decreased in the obese group. Additionally, AMPK analysis parameters (expression, protein level, and activity) did not increase in the obese group. These findings correlated with the partial restoration of mitochondrial function in the obese group, increasing the capacity to maintain the membrane potential after adding calcium as a stressor, and increasing the transversal organization level of the mitochondria analyzed in isolated fibers. SIGNIFICANCE: These results support the notion that obese rats subjected to PA maintain mitochondrial function through mitochondrial fusion activation by an AMPK-independent mechanism.

Analysis of mitochondrial enzymatic activity in blood lymphomonocyte fractions during infection with different Trypanosoma cruzi strains.

Proinflammatory and inflammatory mediators induced by Trypanosoma cruzi infection increase the oxidative stress, generating toxicity for cells targeting mitochondria of different tissues. We studied the activity of citrate synthase and complexes I-IV of respiratory chain in mitochondria of blood lymphomonocyte fraction, from albino Swiss mice infected with different isolates of T. cruzi , during Chagas disease evolution. Complexes I-IV were modified in infected groups (p<0.05) in all the stages, and an inflammatory process of different magnitudes was detected in the heart and skeletal muscle according to the isolate. The citrate synthase activity presented modifications in the SGO Z12 and the Tulahuen group (p<0.05). Hearts showed fiber fragmentation and fibrosis; skeletal muscle presented inflammatory infiltrates and in the Tulahuen infected group, there were also amastigote nests. The inflammatory processes produced an oxidative stress that induced different alterations of mitochondrial enzymes activities in the lymphomonocyte fraction that can be detected by a simple blood extraction, suggesting that they could be used as disease markers, especially in the indeterminate phase of Chagas disease.

Subcellular fractionation of frozen skeletal muscle samples.

Cell fractionation can be used to determine the localization and trafficking of proteins between cellular compartments such as the cytosol, mitochondria, and nuclei. Subcellular fractionation is usually performed immediately after tissue dissection because freezing may fragment cell membranes and induce organellar cross-contamination. Mitochondria are especially sensitive to freezing/thawing and mechanical homogenization. We proposed a protocol to improve the retention of soluble proteins in the mitochondrial fraction obtained from small amounts of frozen skeletal muscle. Fifty milligrams of the red portion of gastrocnemius muscle from Wistar rats were immediately processed or frozen in liquid nitrogen and stored at -80 °C for further processing. We compared the enrichment of subcellular fractions from frozen/fresh samples obtained with the modified protocol with those obtained by standard fractionation. Western blot analyses of marker proteins for cytosolic (alpha-tubulin), mitochondrial (VDAC1), and nuclear (histone-H3) fractions indicated that all of the procedures resulted in enriched subcellular fractions with minimal organellar cross-contamination. Notably, the activity of the soluble protein citrate synthase was higher in the mitochondrial fractions obtained with the modified protocol from frozen/fresh samples compared with the standard protocol. Therefore, our protocol improved the retention of soluble proteins in the mitochondrial fraction and may be suitable for subcellular fractionation of small amounts of frozen skeletal muscle samples.

Twice-a-day training improves mitochondrial efficiency, but not mitochondrial biogenesis, compared with once-daily training.

Exercise training performed with lowered muscle glycogen stores can amplify adaptations related to oxidative metabolism, but it is not known if this is affected by the "train-low" strategy used (i.e., once-daily versus twice-a-day training). Fifteen healthy men performed 3 wk of an endurance exercise (100-min) followed by a high-intensity interval exercise 2 (twice-a-day group, n = 8) or 14 h (once-daily group, n = 7) later; therefore, the second training session always started with low muscle glycogen in both groups. Mitochondrial efficiency (state 4 respiration) was improved only for the twice-a-day group (group × training interaction, P < 0.05). However, muscle citrate synthase activity, mitochondria, and lipid area in intermyofibrillar and subsarcolemmal regions, and PGC1α, PPARα, and electron transport chain relative protein abundance were not altered with training in either group (P > 0.05). Markers of aerobic fitness (e.g., peak oxygen uptake) were increased, and plasma lactate, O2 cost, and rating of perceived exertion during a 100-min exercise task were reduced in both groups, although the reduction in rating of perceived exertion was larger in the twice-a-day group (group × time × training interaction, P < 0.05). These findings suggest similar training adaptations with both training low approaches; however, improvements in mitochondrial efficiency and perceived effort seem to be more pronounced with twice-a-day training.NEW & NOTEWORTHY We assessed, for the first time, the differences between two "train-low" strategies (once-daily and twice-a-day) in terms of training-induced molecular, functional, and morphological adaptations. We found that both strategies had similar molecular and morphological adaptations; however, only the twice-a-day strategy increased mitochondrial efficiency and had a superior reduction in the rating of perceived exertion during a constant-load exercise compared with once-daily training. Our findings provide novel insights into skeletal muscle adaptations using the "train-low" strategy.

Assessment of muscular energy metabolism and heat shock response of the green abalone Haliotis fulgens (Gastropoda: Philipi) at extreme temperatures combined with acute hypoxia and hypercapnia.

The interaction between ocean warming, hypoxia and hypercapnia, suggested by climate projections, may push an organism earlier to the limits of its thermal tolerance window. In a previous study on juveniles of green abalone (Haliotis fulgens), combined exposure to hypoxia and hypercapnia during heat stress induced a lowered critical thermal maximum (CTmax), indicated by constrained oxygen consumption, muscular spams and loss of attachment. Thus, the present study investigated the cell physiology in foot muscle of H. fulgens juveniles exposed to acute warming (18 °C to 32 °C at +3 °C day-1) under hypoxia (50% air saturation) and hypercapnia (~1000 µatm PCO2), alone and in combination, to decipher the mechanisms leading to functional loss in this tissue. Under exposure to either hypoxia or hypercapnia, citrate synthase (CS) activity decreased with initial warming, in line with thermal compensation, but returned to control levels at 32 °C. The anaerobic enzymes lactate and tauropine dehydrogenase increased only under hypoxia at 32 °C. Under the combined treatment, CS overcame thermal compensation and remained stable overall, indicating active mitochondrial regulation under these conditions. Limited accumulation of anaerobic metabolites indicates unchanged mode of energy production. In all treatments, upregulation of Hsp70 mRNA was observed already at 30 °C. However, lack of evidence for Hsp70 protein accumulation provides only limited support to thermal denaturation of proteins. We conclude that under combined hypoxia and hypercapnia, metabolic depression allowed the H. fulgens musculature to retain an aerobic mode of metabolism in response to warming but may have contributed to functional loss.

Hepatic mitochondrial function in Hereford steers with divergent residual feed intake phenotypes.

Variations in phenotypic expression of feed efficiency could be associated with differences or inefficiencies in mitochondria function due to its impact on energy expenditure. The aim of this study was to determine hepatic mitochondrial density and function in terms of respiration, gene and protein expression, and enzyme activity of mitochondrial respiratory complex proteins, in steers of divergent residual feed intake (RFI) phenotypes. Hereford steers (n = 111 and n = 122 for year 1 and 2, respectively) were evaluated in postweaning 70 d standard test for RFI. Forty-six steers exhibiting the greatest (n = 9 and 16 for year 1 and 2; high-RFI) and the lowest (n = 9 and 12 for year 1 and 2; low-RFI) RFI values were selected for this study. After the test, steers were managed together until slaughter under grazing conditions until they reached the slaughter body weight. At slaughter, hepatic samples (biopsies) were obtained. Tissue respiration was evaluated using high-resolution respirometry methods. Data were analyzed using a mixed model that included RFI group as fixed effect and slaughter date and year as a random effect using PROC MIXED of SAS. RFI and dry matter intake were different (P < 0.001) between low and high-RFI groups of year 1 and year 2. Basal respiration and maximum respiratory rate were greater (P ≤ 0.04) for low than high-RFI steers when complex II substrates (succinate) were supplied. However, when Complex I substrates (glutamate/malate) were used maximum respiratory capacity tended to be greater (P < 0.09) for low vs. high-RFI steers. Low-RFI steers presented greater mitochondria density markers (greater (P < 0.05) citrate synthase (CS) activity and tended (P ≤ 0.08) to have greater CS mRNA and mtDNA:nDNA ratio) than high-RFI steers. Hepatic expression SDHA, UQCRC1, and CYC1 mRNA was greater (P ≤ 0.02) and expression of NDUFA4, NDUFA13, SDHD, UQCRH, and ATP5E mRNA tended (P ≤ 0.10) to be greater in low than high-RFI steers. Hepatic SDHA protein expression tended (P < 0.08) to be greater while succinate dehydrogenase activity was greater (P = 0.04) and NADH dehydrogenase activity was greater (P = 0.03) for low than high-RFI steers. High-efficiency steers (low-RFI) probably had greater efficiency in hepatic nutrient metabolism, which was strongly associated with greater hepatic mitochondrial density and functioning, mainly of mitochondrial complex II.

Underlying mechanism of the contractile dysfunction in atrophied ventricular myocytes from a murine model of hypothyroidism.

Hypothyroidism (Hypo) is a risk factor for cardiovascular diseases, including heart failure. Hypo rapidly induces Ca2+ mishandling and contractile dysfunction (CD), as well as atrophy and ventricular myocytes (VM) remodeling. Hypo decreases SERCA-to-phospholamban ratio (SERCA/PLB), and thereby contributes to CD. Nevertheless, detailed spatial and temporal Ca2+ cycling characterization in VM is missing, and contribution of other structural and functional changes to the mechanism underlying Ca2+ mishandling and CD, as transverse tubules (T-T) remodeling, mitochondrial density (Dmit) and energy availability, is unclear. Therefore, in a rat model of Hypo, we aimed to characterize systolic and diastolic Ca2+ signaling, T-T remodeling, Dmit, citrate synthase (CS) activity and high-energy phosphate metabolites (ATP and phosphocreatine). We confirmed a decrease in SERCA/PLB (59%), which slowed SERCA activity (48%), reduced SR Ca2+ (19%) and blunted Ca2+ transient amplitude (41%). Moreover, assessing the rate of SR Ca2+ release (dRel/dt), we found that early and maximum dRel/dt decreased, and this correlated with staggered Ca2+ transients. However, dRel/dt persisted during Ca2+ transient relaxation due to abundant late Ca2+ sparks. Isoproterenol significantly up-regulated systolic Ca2+ cycling. T-T were unchanged, hence, cannot explain staggered Ca2+ transients and altered dRel/dt. Therefore, we suggest that these might be caused by RyR2 clusters desynchronization, due to diminished Ca2+-dependent sensitivity of RyR2, which also caused a decrease in diastolic SR Ca2+ leak. Furthermore, Dmit was unchanged and CS activity slightly decreased (14%), however, the ratio phosphocreatine/ATP did not change, therefore, energy deficiency cannot account for Ca2+ and contractility dysregulation. We conclude that decreased SR Ca2+, due to slower SERCA, disrupts systolic RyR2 synchronization, and this underlies CD.

Perinatal Asphyxia and Brain Development: Mitochondrial Damage Without Anatomical or Cellular Losses.

Perinatal asphyxia remains a significant cause of neonatal mortality and is associated with long-term neurodegenerative disorders. In the present study, we evaluated cellular and subcellular damages to brain development in a model of mild perinatal asphyxia. Survival rate in the experimental group was 67%. One hour after the insult, intraperitoneally injected Evans blue could be detected in the fetuses' brains, indicating disruption of the blood-brain barrier. Although brain mass and absolute cell numbers (neurons and non-neurons) were not reduced after perinatal asphyxia immediately and in late brain development, subcellular alterations were detected. Cortical oxygen consumption increased immediately after asphyxia, and remained high up to 7 days, returning to normal levels after 14 days. We observed an increased resistance to mitochondrial membrane permeability transition, and calcium buffering capacity in asphyxiated animals from birth to 14 days after the insult. In contrast to ex vivo data, mitochondrial oxygen consumption in primary cell cultures of neurons and astrocytes was not altered after 1% hypoxia. Taken together, our results demonstrate that although newborns were viable and apparently healthy, brain development is subcellularly altered by perinatal asphyxia. Our findings place the neonate brain mitochondria as a potential target for therapeutic protective interventions.