Changes In Oxidative Metabolism Research Articles

Adaptogenic correction of free radical processes in liver tissue under the exposure to coal-rock dust on the body (experimental study)

Introduction. Damage to the liver under the conditions of long-term exposure to coal-rock dust on the body leads to significant impairment of its metabolic, detoxification, synthetic, and other functions. In this regard, it is relevant to search for early methods of prevention and correction of developing pathological changes in this organ. The purpose of the study is to experimentally study the effect of a complex drug with dihydroquercetin on the activity of free radical processes in the liver of laboratory rats over the dynamics of long-term exposure to coal-rock dust. Materials and methods. The work was carried out on male white rats weighing 200–250 g. The control group was kept under the standard vivarium conditions. The experimental group included rats that inhaled coal-rock dust in a priming chamber daily for 4 hours for 1, 3, 6, and 9 weeks and rats that received a daily complex drug with dihydroquercetin immediately before dust exposure. In the liver tissue, the activity of free radical oxidation and antioxidant defense enzymes (superoxide dismutase and catalase) was determined. Results. Priming rats with coal-rock dust for three weeks led to the gain in the intensity of free radical processes in the liver tissue: the initial level of accumulation of TBA-active oxidation products reliably raised by almost 2 times over the 1st week and 3 times over the 3rd week of the experiment compared to the control. An increase in the intensity of free radical oxidation was accompanied by activation of antioxidant defense enzymes - catalase - by 2.4 times at the 1st week and 1.6 times at the 3rd week of dust exposure. Raising the period of priming with dust up to 9 weeks led to multidirectional changes in oxidative metabolism in the liver tissue: at week 6, a decrease in the activity of superoxide dismutase was almost 3 times; at week 9, to a slight activation of antioxidant enzymes: superoxide dismutase by 55% and catalase by 36%.The most effective use of a complex drug with dihydroquercetin had been shown in short-term mode for three weeks, but not long-term (more than 6 weeks). The maximum antioxidant defense effect of dihydroquercetin was detected at the 3rd week of dust exposure – the level of free radical products decreased by 2 times and the activity of superoxide dismutase increased by 4 times. Limitations. Since the issue of organ-specific effects and the optimal duration of use of dihydroquercetin to increase resistance to free radical damage has been little elaborated, this imposes certain restrictions on its use for the correction and prevention of occupational diseases. Conclusion. The results obtained should be taken into account with long-term use of exogenous antioxidants in the correction and prevention of occupational diseases to support the level of endogenous antioxidant systems in the cell and the protective effect of dihydroquercetin for tissues most sensitive to the action of free radical processes.

The bisphosphonates alendronate and zoledronate induce adaptations of aerobic metabolism in permanent human endothelial cells

Nitrogen-containing bisphosphonates (NBPs), compounds that are widely used in the treatment of bone disorders, may cause side effects related to endothelial dysfunction. The aim of our study was to investigate the effects of chronic 6-day exposure to two common bone-preserving drugs, alendronate and zoledronate, on endothelial function and oxidative metabolism of cultured human endothelial cells (EA.hy926). NBPs reduced cell viability, induced oxidative stress and a pro-inflammatory state and downregulated the prenylation-dependent ERK1/2 signaling pathway in endothelial cells. In addition, NBPs induced increased anaerobic respiration and slightly increased oxidative mitochondrial capacity, affecting mitochondrial turnover through reduced mitochondrial fission. Moreover, by blocking the mevalonate pathway, NBPs caused a significant decrease in the level of coenzyme Q10, thereby depriving endothelial cells of an important antioxidant and mitochondrial electron carrier. This resulted in increased formation of reactive oxygen species (ROS), upregulation of antioxidant enzymes, and impairment of mitochondrial respiratory function. A general decrease in mitochondrial respiration occurred with stronger reducing fuels (pyruvate and glutamate) in NBP-treated intact endothelial cells, and significantly reduced phosphorylating respiration was observed during the oxidation of succinate and especially malate in NBP-treated permeabilized endothelial cells. The observed changes in oxidative metabolism caused a decrease in ATP levels and an increase in oxygen levels in NBP-treated cells. Thus, NBPs modulate the energy metabolism of endothelial cells, leading to alterations in the cellular energy state, coenzyme Q10 redox balance, mitochondrial respiratory function, and mitochondrial turnover.

Impacto de dos tratamientos hidrotérmicos aplicados secuencialmente sobre el metabolismo oxidativo relacionado con la tolerancia al daño por frío en frutos de mango

Mango is susceptible to chilling injury (CI). Its short-term exposure to heat treatments can induce tolerance to this disorder; however, a short time is not enough to control the fruit fly. In this sense, the application of sequential heat treatments where the quarantine treatment is included can bring great benefits for mango marketing. The aim of this research was to evaluate the effect of two different sequentially-applied hot water treatments (HWT) on CI tolerance and changes in oxidative metabolism in mango. Mangoes were divided into four groups: control, HWT1 (dipping in water at 46.1 °C for 75 min), HWT2 (dipping in water at 55 °C for 5 min), and HWT1 + HWT2. Fruits were stored at 5 °C/30 days to induce CI and thereafter at 21 °C for 8 days. Three replicates with five repetitions were used to evaluate CI index, electrolyte leakage (EL), malondialdehyde (MDA), weight loss (WL), color, firmness and enzymatic antioxidant activity. The use of HWT1 reduced CI during low temperature storage, while HWT2 and the HWT1 + HWT2 showed lower CI symptoms during ripening. The use of HWT1 showed a similar EL % and higher MDA content (7.07 x 104 nmol·L-1) than the control (6.68 x 104 nmol·L-1). HWT1 + HWT2 showed the lowest WL (2.98 %) and the highest enzymatic antioxidant activity during cold storage and during the first days at 21 °C, while HWT2 showed the highest color and firmness retention. Hot water treatments applied sequentially can be considered an effective alternative to induce CI tolerance and to maintain the quality of mango.

ВЛИЯНИЕ НИЗКОИНТЕНСИВНОГО ЭЛЕКТОРМАГНИТНОГО ИЗЛУЧЕНИЯ КРАЙНЕ ВЫСОКОЙ ЧАСТОТЫ НА ПОКАЗАТЕЛИ ТКАНЕВОГО ОКИСЛИТЕЛЬНОГО МЕТАБОЛИЗМА

The paper considers the indicators of tissue oxidative in rats after 10-fold exposure to low-intensity electromagnetic radiation of the millimeter range. The experiment was carried out on 40 mature male rats of the Wistar line weighing 200-220 g, which were kept in a vivarium with a natural light regime. The animals were divided into 2 groups of 20 rats each. The animals in the first group were biological controls and were in normal vivarium conditions, the animals of the second group were exposed to EHF EMR for 10 sessions daily, in the morning. On the 10th day of EHF exposure, tissue fluorescence indicators were recorded by the diagnostic complex “LAZMA MC” (Modification of LAZMA-MC-III, LAZMA, Moscow, Russia). The results of the study showed that rats exposed to 10-fold EHF exposure had an increase in the values of normalized NADH fluorescence amplitudes and an increase in FAD amplitudes by 82.5% (p<0.05) and 42.6% (p<0.05), as well as redox potential - by 36.9% (p<0.05), respectively, compared to with control. These changes indicate that 10-fold exposure to low-intensity EHF EMR causes an increase in the contribution of aerobic metabolism in rats mainly due to oxidative phosphorylation, leads to activation of the respiratory chain. It can be assumed that these changes in oxidative metabolism are determined primarily by the process of adaptation of microcirculatory blood flow to the needs of tissues for nutrients and the removal of metabolites.

The gut-microbiota-brain changes across the liver disease spectrum.

Gut microbiota dysbiosis plays a significant role in the progression of liver disease, and no effective drugs are available for the full spectrum. In this study, we aimed to explore the dynamic changes of gut microbiota along the liver disease spectrum, together with the changes in cognition and brain metabolism. Sprague–Dawley rats were divided into four groups reflecting different stages of liver disease: control diet (NC); high-fat, high-cholesterol diet (HFHC), emulating non-alcoholic steatohepatitis; control diet + thioacetamide (NC + TAA), simulating acute liver failure; and high-fat, high-cholesterol diet + thioacetamide (HFHC + TAA) to assess the effect of the superimposed damages. The diet was administered for 14 weeks and the thioacetamide was administrated (100 mg/kg day) intraperitoneally over 3 days. Our results showed changes in plasma biochemistry and liver damage across the spectrum. Differences in gut microbiota at the compositional level were found among the experimental groups. Members of the Enterobacteriaceae family were most abundant in HFHC and HFHC + TAA groups, and Akkermansiaceae in the NC + TAA group, albeit lactobacilli genus being dominant in the NC group. Moreover, harm to the liver affected the diversity and bacterial community structure, with a loss of rare species. Indeed, the superimposed damage group (HFHC + TAA) suffered a loss of both rare and abundant species. Behavioral evaluation has shown that HFHC, NC + TAA, and HFHC + TAA displayed a worsened execution when discriminating the new object. Also, NC + TAA and HFHC + TAA were not capable of recognizing the changes in place of the object. Furthermore, working memory was affected in HFHC and HFHC + TAA groups, whereas the NC + TAA group displayed a significant delay in the acquisition. Brain oxidative metabolism changes were observed in the prefrontal, retrosplenial, and perirhinal cortices, as well as the amygdala and mammillary bodies. Besides, groups administered with thioacetamide presented an increased oxidative metabolic activity in the adrenal glands. These results highlight the importance of cross-comparison along the liver spectrum to understand the different gut–microbiota–brain changes. Furthermore, our data point out specific gut microbiota targets to design more effective treatments, though the liver–gut–brain axis focused on specific stages of liver disease.

Interplay between mitochondrial reactive oxygen species, oxidative stress and hypoxic adaptation in facioscapulohumeral muscular dystrophy: Metabolic stress as potential therapeutic target

Facioscapulohumeral muscular dystrophy (FSHD) is characterised by descending skeletal muscle weakness and wasting. FSHD is caused by mis-expression of the transcription factor DUX4, which is linked to oxidative stress, a condition especially detrimental to skeletal muscle with its high metabolic activity and energy demands. Oxidative damage characterises FSHD and recent work suggests metabolic dysfunction and perturbed hypoxia signalling as novel pathomechanisms. However, redox biology of FSHD remains poorly understood, and integrating the complex dynamics of DUX4-induced metabolic changes is lacking.Here we pinpoint the kinetic involvement of altered mitochondrial ROS metabolism and impaired mitochondrial function in aetiology of oxidative stress in FSHD. Transcriptomic analysis in FSHD muscle biopsies reveals strong enrichment for pathways involved in mitochondrial complex I assembly, nitrogen metabolism, oxidative stress response and hypoxia signalling. We found elevated mitochondrial ROS (mitoROS) levels correlate with increases in steady-state mitochondrial membrane potential in FSHD myogenic cells. DUX4 triggers mitochondrial membrane polarisation prior to oxidative stress generation and apoptosis through mitoROS, and affects mitochondrial health through lipid peroxidation. We identify complex I as the primary target for DUX4-induced mitochondrial dysfunction, with strong correlation between complex I-linked respiration and cellular oxygenation/hypoxia signalling activity in environmental hypoxia. Thus, FSHD myogenesis is uniquely susceptible to hypoxia-induced oxidative stress as a consequence of metabolic mis-adaptation. Importantly, mitochondria-targeted antioxidants rescue FSHD pathology more effectively than conventional antioxidants, highlighting the central involvement of disturbed mitochondrial ROS metabolism. This work provides a pathomechanistic model by which DUX4-induced changes in oxidative metabolism impair muscle function in FSHD, amplified when metabolic adaptation to varying O2 tension is required.

Transcriptomic analysis elucidates the enhanced skeletal muscle mass, reduced fat accumulation, and metabolically benign liver in human follistatin-344 transgenic pigs

Follistatin (FST) is an important regulator of skeletal muscle growth and adipose deposition through its ability to bind to several members of the transforming growth factor-β (TGF-β) superfamily, and thus may be a good candidate for future animal breeding programs. However, the molecular mechanisms underlying the phenotypic changes have yet to be clarified in pig. We generated transgenic (TG) pigs that express human FST specifically in skeletal muscle tissues and characterized the phenotypic changes compared with the same tissues in wild-type pigs. The TG pigs showed increased skeletal muscle growth, decreased adipose deposition, and improved metabolism status (P<0.05). Transcriptome analysis detected important roles of the PIK3–AKT signaling pathway, calcium-mediated signaling pathway, and amino acid metabolism pathway in FST-induced skeletal muscle hypertrophy, and depot-specific oxidative metabolism changes in psoas major muscle. Furthermore, the lipid metabolism-related process was changed in adipose tissue in the TG pigs. Gene set enrichment analysis revealed that genes related to lipid synthesis, lipid catabolism, and lipid storage were down-regulated (P<0.01) in the TG pigs for subcutaneous fat, whereas genes related to lipid catabolism were significantly up-regulated (P<0.05) in the TG pigs for retroperitoneal fat compared with their expression levels in wild-type pigs. In liver, genes related to the TGF-β signaling pathway were over-represented in the TG pigs, which is consistent with the inhibitory role of FST in regulating TGF-β signaling. Together, these results provide new insights into the molecular mechanisms underlying the phenotypic changes in pig.

A High Fat/Cholesterol Diet Recapitulates Some Alzheimer's Disease-Like Features in Mice: Focus on Hippocampal Mitochondrial Dysfunction.

Ample evidence from clinical and pre-clinical studies suggests mid-life hypercholesterolemia as a risk factor for developing Alzheimer's disease (AD) at a later age. Hypercholesterolemia induced by dietary habits can lead to vascular perturbations that increase the risk of developing sporadic AD. To investigate the effects of a high fat/cholesterol diet (HFCD) as a risk factor for AD by using a rodent model of AD and its correspondent control (healthy animals). We compared the effect of a HFCD in normal mice (non-transgenic mice, NTg) and the triple transgenic mouse model of AD (3xTgAD). We evaluated cognitive performance in relation to changes in oxidative metabolism and neuron-derived nitric oxide (•NO) concentration dynamics in hippocampal slices as well as histochemical staining of markers of the neurovascular unit. In NTg, the HFCD produced only moderate hypercholesterolemia but significant decline in spatial memory was observed. A tendency for decrease in •NO production was accompanied by compromised mitochondrial function with decrease in spare respiratory capacity. In 3xTgAD mice, a robust increase in plasma cholesterol levels with the HFCD did not worsen cognitive performance but did induce compromise of mitochondrial function and significantly decreased •NO production. We found increased staining of biomarkers for astrocyte endfeet and endothelial cells in 3xTgAD hippocampi, which was further increased by the HFCD. A short term (8 weeks) intervention with HFCD can produce an AD-like phenotype even in the absence of overt systemic hypercholesterolemia and highlights mitochondrial dysfunction as a link between hypercholesterolemia and sporadic AD.

MiR-373 Suppresses Cell Proliferation and Apoptosis via Regulation of SIRT1/PGC-1α/NRF2 Axis in Pancreatic Cancer.

Objective Our study aimed to investigate function and mechanism of miR-373 in proliferation and apoptosis of pancreatic cancer (PC) cells by regulating NAD+-dependent histone deacetylase sirtulin 1 (SIRT1). Materials and Methods This experimental study included two PC cell lines AsPC-1 and PANC-1 in which expression levels of miR-373 and SIRT1 were manipulated. The level of miR-373 was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. Expression levels of SIRT1, BCL-2, BAX, cleaved CASPASE-8/9/3, PARP, PGC-1α, NRF2, eNOS and iNOS were examined via RT-qPCR and western blotting, respectively. The binding sites of miR-373 on the SIRT1 were examined via dual-luciferase assay. Cell proliferation and apoptosis were examined by MTT assay, colony formation assay, Annexin-V/PI staining and TUNEL assay. The oxidative metabolic changes were monitored by reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) detection. Results miR-373 could specifically target the 3’-UTR of SIRT1 and reduce its expression in PC cells. Either elevated expression of miR-373 or partial loss of SIRT1 inhibited cell proliferation and induced cell apoptosis. Accumulation of BAX and cleaved CASPASE-8/9/3, inhibition of PGC-1α/NRF2 pathway, increase oxidative stress and reduction of BCL-2 as well as uncleaved PARP were found in the presence of miR-373 or the absence of SIRT1. Overexpression of SIRT1 could reduce anti-proliferative and pro-apoptotic effects of miR-373. ConclusionOverall, this study concluded that miR-373-dependent SIRT1 inhibition displays anti-proliferative and pro- apoptotic roles in PC cells via PGC-1α/NRF2 pathway, which highlights miR-373 as a potential target for PC treatment.

Changes of oxidative metabolism in the roots of wheat (Triticum aestivum L.) seedlings in response to elevated ammonium concentrations

To elucidate the response of oxidative metabolism, triggered by elevated ammonium (NH4+) concentrations, on root growth of wheat seedlings, Yumai 49 (NH4+-tolerant) and Lumai 15 (NH4+-sensitive) cultivars were supplied with either 5.0 mmol L−1 NH4+-N (EAC) or 5.0 mmol L−1 NO3–-N (CON) under hydroponic conditions. Root growth in both cultivars was significantly reduced under EAC, and the negative effect was greater in Lumai 15. EAC enhanced the activities of monodehydroascorbate reductase and dehydroascorbate reductase in the roots of both cultivars, while it decreased ascorbic acid (ASA) content and GDP-mannose pyrophosphorylase (GMPase) activity at the 12th day after treatment in Lumai 15 by 62.0 and 71.4%; and in Yumai 49 by 38.8 and 62.2%, respectively, indicating that the regeneration of ASA was increased, but the biosynthesis of ASA was reduced under EAC treatment. Moreover, EAC increased DHA/ASA, reactive oxygen species (ROS), and malondialdehyde contents, as well as antioxidant enzyme activities in the roots of both cultivars. Relatively greater increases in ROS and soluble sugar, and lower antioxidant enzyme activities in Lumai 15 indicate severe disruption of oxidative metabolism when compared to Yumai 49. Results reveal that the reduction of ASA biosynthesis via decreased GMPase activity under the EAC condition probably acts as a trigger for accumulated ROS and imbalanced redox status, resulting in root growth inhibition during wheat seedling growth stage. Yumai 49, being an NH4+ -tolerant cultivar, had the stronger capacity to protect itself from oxidative stress, which allowed it to retain a lower DHA to ASA ratio by maintaining a better redox homeostasis than could be maintained in the NH4+ -sensitive cultivar Lumai 15.

Triphenylphosphonium derivatives disrupt metabolism and inhibit melanoma growth in vivo when delivered via a thermosensitive hydrogel.

Despite dramatic improvements in outcomes arising from the introduction of targeted therapies and immunotherapies, metastatic melanoma is a highly resistant form of cancer with 5 year survival rates of <35%. Drug resistance is frequently reported to be associated with changes in oxidative metabolism that lead to malignancy that is non-responsive to current treatments. The current report demonstrates that triphenylphosphonium(TPP)-based lipophilic cations can be utilized to induce cytotoxicity in pre-clinical models of malignant melanoma by disrupting mitochondrial metabolism. In vitro experiments demonstrated that TPP-derivatives modified with aliphatic side chains accumulated in melanoma cell mitochondria; disrupted mitochondrial metabolism; led to increases in steady-state levels of reactive oxygen species; decreased total glutathione; increased the fraction of glutathione disulfide; and caused cell killing by a thiol-dependent process that could be rescued by N-acetylcysteine. Furthermore, TPP-derivative-induced melanoma toxicity was enhanced by glutathione depletion (using buthionine sulfoximine) as well as inhibition of thioredoxin reductase (using auranofin). In addition, there was a structure-activity relationship between the aliphatic side-chain length of TPP-derivatives (5–16 carbons), where longer carbon chains increased melanoma cell metabolic disruption and cell killing. In vivo bio-distribution experiments showed that intratumoral administration of a C14-TPP-derivative (12-carbon aliphatic chain), using a slow-release thermosensitive hydrogel as a delivery vehicle, localized the drug at the melanoma tumor site. There, it was observed to persist and decrease the growth rate of melanoma tumors. These results demonstrate that TPP-derivatives selectively induce thiol-dependent metabolic oxidative stress and cell killing in malignant melanoma and support the hypothesis that a hydrogel-based TPP-derivative delivery system could represent a therapeutic drug-delivery strategy for melanoma.

Contribution of thermogenic mechanisms by male and female mice lacking pituitary adenylate cyclase-activating polypeptide in response to cold acclimation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide critical to the regulation of the stress response, including having a role in energy homeostasis. Mice lacking PACAP are cold-sensitive and have impaired adrenergic-induced thermogenesis. Interestingly, Pacap null mice can survive cold housing if acclimated slowly, similar to observations in uncoupling protein 1 (UCP1)-deficient mice. We hypothesized that Pacap null mice use alternate thermogenic pathways to compensate for impaired adaptive thermogenesis when acclimated to cold. Observations of behavior and assessment of fiber type in skeletal muscles did not show evidence of prolonged burst shivering or changes in oxidative metabolism in male or female Pacap-/- mice during cold acclimation compared with Pacap+/+ mice. Despite previous work that has established impaired capacity for adaptive thermogenesis in Pacap null mice, adaptive thermogenesis can be induced in mice lacking PACAP to support survival with cold housing. Interestingly, sex-specific morphological and molecular differences in adipose tissue remodeling were observed in Pacap null mice compared with controls. Thus, sexual dimorphisms are highlighted in adipose tissue remodeling and thermogenesis with cold acclimation in the absence of PACAP.NEW & NOTEWORTHY This manuscript adds to the literature of endocrine regulation of adaptive thermogenesis and energy balance. It specifically describes the role of pituitary adenylate cyclase-activating polypeptide on the regulation of brown adipose tissue via the sympathetic nervous system with a focus on compensatory mechanisms of thermogenesis. We highlight sex-specific differences in energy metabolism.

Characterization of Seasonal Pharmacokinetic Variability in Woodchucks.

The eastern woodchuck (Marmota monax) is a hibernating species extensively used as an in vivo efficacy model for chronic human hepatitis B virus infection. Under laboratory conditions, woodchucks develop a pseudohibernation condition; thus, the pharmacokinetics (PK) of small-molecule therapeutics may be affected by the seasonal change. The seasonal PK of four probe compounds were characterized over 12 months in seven male and nine female laboratory-maintained woodchucks. These compounds were selected to study changes in oxidative metabolism [antipyrine (AP)], glucuronidation [raltegravir (RTG)], renal clearance [lamivudine (3TC)], and hepatic function [indocyanine green (ICG)]. Seasonal changes in physiologic parameters and PK were determined. Seasonal body weight increases were ≥30%. Seasonal changes in body temperature and heart rate were <10%. The mean AP exposure remained unchanged from April to August 2017, followed by a significant increase (≥1.0-fold) from August to December and subsequent decrease to baseline at the end of study. A similar trend was observed in RTG and 3TC exposures. The ICG exposure remained unchanged. No significant sex difference in PK was observed, although female woodchucks appeared to be less susceptible to seasonal PK and body weight changes. Significant seasonal PK changes for AP, RTG, and 3TC indicate decreases in oxidative metabolism, phase II glucuronidation, and renal clearance during pseudohibernation. The lack of seasonal change in ICG exposure suggests there are no significant changes in hepatic function. This information can be used to optimize the scheduling of woodchuck studies to avoid seasonally driven variation in drug PK. SIGNIFICANCE STATEMENT: Woodchuck is a hibernating species and is commonly used as a nonclinical model of hepatitis B infection. Investigation of seasonal PK changes is perhaps of greater interest to pharmaceutical industry scientists, who use the woodchuck model to optimize the scheduling of woodchuck studies to avoid seasonally driven variation in drug PK and/or toxicity. This information is also valuable to drug metabolism and veterinary scientists in understanding woodchuck's seasonal metabolism and behavior under the pseudohibernation condition.

The Level of Reactive Carbonyl Derivatives of Proteins, Methylglyoxal, and Malondialdehyde in Rats Experiencing Chronic Unpredictable Moderate Stress

BACKGROUND: Prolonged exposure to stress is known to change homeostasis and can lead to the development of psychopathological disorders, including depression. Exposure to stress factors contributes to the emergence of reactive oxygen species in the brain. The data characterizing changes of the oxidative metabolism under conditions of chronic stress are insufficient, which determine the relevance of the research in this direction. The determination of reactive carbonyl derivatives of proteins (RCDP), methylglyoxal (MG), and malondialdehyde (MDA) levels can be considered an informative method for studying the dynamics of oxidative metabolism.&#x0D; AIM: The aim of the study was to study the RCDP, MG, and MDA in red blood cells and blood plasma in rats experiencing chronic unpredictable moderate stress (CUMS).&#x0D; METHODS: A total of 20 male outbred rats weighing 450–500 g were used in the study. The animals were divided into two groups of ten rats each using randomized selection. The experimental group of animals was exposed to the impact of diverse stress factors, according to the CUMS model for 21 days. The control group of animals remained in standard conditions and did not undergo CUMS model. Blood sampling was performed twice for all rats the day before the experiment started and on 21st day to determine the level of RCDP, MG, and MDA in their erythrocytes and blood plasma.&#x0D; RESULTS: The study revealed a significant decrease of RCDP (p = 0.007) and MDA levels in erythrocytes (p = 0.013), and MDA level in blood plasma (p = 0.005) of rats of the main experimental group. The control group showed a significant increase of RCDP level in plasma (p = 0.008), a two-fold increase of MDA in erythrocytes (p = 0.05) with a decrease of MDA in plasma (p = 0.011). A tendency for MG increase was noted in erythrocytes of rats in both the main group and the control group.&#x0D; CONCLUSION: The early stages of CUMS were accompanied by the violation of oxidative metabolism with the emergence and accumulation of RCDP and MDA in the blood of animals. The decreased emergence of RCDP and MDA in the later stages can be associated with an excess of oxidized substrates in the blood of animals of the experimental group. The revealed trend toward MG growth in animals of both groups suggests its increased synthesis in erythrocytes, which, in our opinion, should be considered a negative factor affecting the deformability of red cells. In turn, a violation of the deformability of red blood cells leads to a violation of microcapillary blood flow, which contributes to the development of hypoxic damage in different tissues. Thus, the obtained data confirm the different directions of changes in RCDP and MDA levels in the blood of animals experiencing CUMS or acute stress. Our results prove the need for further research on changes in oxidative metabolism in chronic stress disorders.

P-379 - Sirtuin- cAMP crosstalk in metabolic diseases: nucleus versus mitochondria

Mitochondrial dysfunction in human pathologies make mitochondrial signaling systems attractive therapeutic targets. Sirtuins are deacylases that play a central role in mitochondrial function, metabolic regulation and aging. Indeed, efficient mitochondria and adaptive changes in oxidative metabolism were shown to be coordinated by signaling pathways, activated by physiological stimuli, controlling an intense crosstalk between nucleus and mitochondrial genomes. In this study, SIRT3 or SIRT1 KO as well as wild-type MEFs were incubated with specific activators and inhibitors controlling these signaling pathways. We observed an improvement of mitochondrial function in SIRT3-/- MEFs and SIRT1-/- MEFs by observing a dose- and time-dependent increase in mitochondrial membrane potential. Moreover, no changes were observed for reactive oxidative species formation and an increase in nuclear-encoded mitochondrial genes were observed. Here, we are starting to understand the crosstalk between two major signaling systems in two cell compartments. Pharmacological or alternative therapies offer promising approaches to target and manipulate these systems leading to the maintenance of mitochondrial content and function, and ultimately prevention of cellular dysfunction.

Mitochondrial Respiratory Capacity and Coupling Control of Skeletal Muscle in Colon-26 Tumor-Induced Cachexia

Cancer cachexia is a life-threatening, paraneoplastic syndrome featuring unintended weight loss and skeletal muscle atrophy. Mitochondria, the major providers of cellular energy, couple oxygen consumption to ATP synthesis (i.e. oxidative phosphorylation, OXPHOS). Impaired mitochondrial bioenergetics (e.g. respiration) is associated with the pathophysiology of multiple diseases. The control of mitochondrial respiration in skeletal muscle during the induction and progression of cancer cachexia is not well understood. PURPOSE: To investigate mitochondrial respiratory capacity and coupling control of skeletal muscle in the colon-26 model of cancer cachexia. METHODS: Balb/c males (10 wks) were assigned to control or colon-26 (C26). C26 mice were injected with 1x106 tumor cells, and tissue collected on days 7, 14, and 21 post-injection. In this model, mice develop palpable tumors at day 7, and cachexia by day 21. Controls were injected with PBS and tissue collected on day 0. Respiration was measured in permeabilized fibers from the medial gastrocnemius via high-resolution respirometry. A substrate-uncoupler-inhibitor titration protocol was used to evaluate Complex I OXPHOS (CIP), Complex I+II OXPHOS (CI+IIP), and electron transfer system capacity (ETS). Efficiency of the OXPHOS system was determined from the ratio CI+IIP/ETS (P/E). RESULTS: CIP was significantly lower (p<0.05) at day 21 (4.8±1.6 pmol/s/mg) in comparison to day 0 (53.4±7.0), day 7 (57.4±6.5), and day 14 (60.0±2.9). CI+IIP was significantly lower (p<0.05) at day 21 (22.3±2.3 pmol/s/mg) in comparison to day 0 (65.5±7.8), day 7 (69.3±8.5), and day 14 (73.8±4.8). Maximal ETS was significantly lower (p<0.05) at day 21 (24.1 pmol/s/mg) in comparison to day 0 (83.7±13.8), day 7 (84.4±12.3), and day 14 (105.1±7.3). P/E was not significantly different across timepoints (p>0.05). CONCLUSION: Phosphorylating respiration with electron input from Complex I and I+II, and maximal electron transfer system capacity (i.e. non-coupled respiration) was significantly reduced at day 21 concomitant with cachexia, but not at earlier timepoints, suggesting that changes in oxidative metabolism occur as a consequence of cachexia rather than having a causative role. Loss of respiratory capacity may compromise muscle function and physical independence.

Cryo-electron microscopy structures and progress toward a dynamic understanding of KATP channels.

Adenosine triphosphate (ATP)-sensitive K+ (KATP) channels are molecular sensors of cell metabolism. These hetero-octameric channels, comprising four inward rectifier K+ channel subunits (Kir6.1 or Kir6.2) and four sulfonylurea receptor (SUR1 or SUR2A/B) subunits, detect metabolic changes via three classes of intracellular adenine nucleotide (ATP/ADP) binding site. One site, located on the Kir subunit, causes inhibition of the channel when ATP or ADP is bound. The other two sites, located on the SUR subunit, excite the channel when bound to Mg nucleotides. In pancreatic β cells, an increase in extracellular glucose causes a change in oxidative metabolism and thus turnover of adenine nucleotides in the cytoplasm. This leads to the closure of KATP channels, which depolarizes the plasma membrane and permits Ca2+ influx and insulin secretion. Many of the molecular details regarding the assembly of the KATP complex, and how changes in nucleotide concentrations affect gating, have recently been uncovered by several single-particle cryo-electron microscopy structures of the pancreatic KATP channel (Kir6.2/SUR1) at near-atomic resolution. Here, the author discusses the detailed picture of excitatory and inhibitory ligand binding to KATP that these structures present and suggests a possible mechanism by which channel activation may proceed from the ligand-binding domains of SUR to the channel pore.

Epigenetic memory of oxidative stress: does nephrilin exert its protective effects via Rac1?

AimNephrilin peptide, a designed inhibitor of Rictor complex (mTORC2), exerts pleiotropic protective effects in metabolic, xenobiotic and traumatic stress models. Stress can generate enduring epigenetic changes in gene function. In this work we examine the possibility that nephrilin treatment protects against acute and enduring global changes in oxidative metabolism, with a focus on the Rictor-complex-mediated activation of Rac1, a subunit of NADPH oxidase (Nox) via PKCs, Prex1 and p66shc.MethodsGiven the wide range of animal models in which nephrilin peptide has previously demonstrated effectiveness in vivo, we chose three different experimental systems for this investigation: dermal fibroblasts, renal proximal tubule epithelial cells (PTECs), and kidney tissue and urine from an animal model of burn trauma in which nephrilin was previously shown to prevent loss of kidney function.Results(1) Nephrilin protects dermal fibroblasts from loss of viability and collagen synthesis after ultraviolet A (UV-A) or H2O2 insult. (2) Nephrilin reduces reactive oxygen species (ROS) formation by H2O2–treated (PTECs) with or without nicotine pretreatment. Using RNA arrays and pathway analysis we demonstrate that nicotine and H2O2-treated PTECs specifically induced Rac1 gene networks in these cells. (3) Using kidney tissue and urine from the burn trauma model we demonstrate significant elevations of [a] 8-aminoprostane in urine; [b] kidney tissue histone modification and DNA methylation; and [c] post-transcriptional phosphorylation events consistent with Rac1 activation in kidney tissue.ConclusionNephrilin protects against oxidative stress, possibly by modulating the activation of Rac1.

Ultra-weak photon emission as a dynamic tool for monitoring oxidative stress metabolism

In recent years, excessive oxidative metabolism has been reported as a critical determinant of pathogenicity in many diseases. The advent of a simple tool that can provide a physiological readout of oxidative stress would be a major step towards monitoring this dynamic process in biological systems, while also improving our understanding of this process. Ultra-weak photon emission (UPE) has been proposed as a potential tool for measuring oxidative processes due to the association between UPE and reactive oxygen species. Here, we used HL-60 cells as an in vitro model to test the potential of using UPE as readout for dynamically monitoring oxidative stress after inducing respiratory burst. In addition, to probe for possible changes in oxidative metabolism, we performed targeted metabolomics on cell extracts and culture medium. Lastly, we tested the effects of treating cells with the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). Our results show that UPE can be used as readout for measuring oxidative stress metabolism and related processes.

Epithelial-mesenchymal transition induction is associated with augmented glucose uptake and lactate production in pancreatic ductal adenocarcinoma.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a common malignancy with dismal prognosis. Metastatic spread and therapeutic resistance, the main causes of PDAC-related mortalities, are both partially underlined by the epithelial-mesenchymal transition (EMT) of PDAC cells. While the role of Warburg metabolism has been recognized in supporting rapid cellular growth and proliferation in many cancer types, less is known about the metabolic changes occurring during EMT, particularly in the context of PDAC.ResultsIn the current study, experimental models of EMT were established in the Panc-1 cell line of human PDAC via exposure to two physiologically relevant EMT inducers (tumor necrosis factor-α and transforming growth factor-β) and the metabolic consequences examined. The two EMT models displayed similar alterations in the general metabolic profile including augmented glucose uptake and lactate secretion as well as the lack of change in oxidative metabolism. Examination of molecular markers revealed differences in the pathways underlying the metabolic rewiring. 13C-Glucose tracer data confirmed that a major portion of accumulated lactate was derived from glucose, but subsequent flux analysis suggested involvement of non-canonical pathways towards lactate production.ConclusionsOur results characterize the metabolic reprogramming occurring during PDAC cell EMT and highlight the common changes of increased glucose uptake and lactate secretion under different EMT conditions. Such insight is urgently required for designing metabolic strategies to selectively target cells undergoing EMT in PDAC.Electronic supplementary materialThe online version of this article (doi:10.1186/s40170-016-0160-x) contains supplementary material, which is available to authorized users.