Purpose: Aging is a complex biological process that begins at the cellular level, disrupting energy homeostasis. This study investigated the role of Sirt3, major mitochondrial deacetylase involved in metabolic pathways, in sex-dependent changes in energy homeostasis during aging in kidney of Sirt3 WT and KO mice.

Methods: Enzymatic activity, lipid peroxidation, protein carbonylation with Western blot and metabolomic analyses were performed to assess physiological and metabolic parameters

Results: Higher Sirt3 expression in male WT mice leads to increased vulnerability to its deficiency, as reflected in the shorter lifespan of male KO mice. This is further supported by distinct metabolomic clustering in male KO mice, highlighting significant metabolic disruptions. Male-specific declines in metabolites such as creatine, phosphorylcholine, trimethylamine-N-oxide, and L-carnitine, along with reduced trifunctional multienzyme complex subunit β (HADHB) expression, point to impaired fatty acid metabolism and mitochondrial dysfunction.

Conclusions: The findings emphasize the sex-specific function of Sirt3 in regulating mitochondrial activity, energy metabolism, and oxidative stress in the murine kidney, with male mice exhibiting a greater reliance on Sirt3 for metabolic stability.

Nitrite is the typical byproduct of nitric oxide (^•NO) autooxidation in biological systems. However, certain circumstances favor its reduction “back” to the signaling free radical, providing a non-enzymatic route for the synthesis of ^•NO. In pathophysiological conditions such as ischemia/reperfusion (I/R), where low oxygen availability limits nitric oxide synthase activity, nitrite reduction to ^•NO may allow protective modulation of mitochondrial oxidative metabolism and thus reduce the impact of I/R on brain tissue. In the current study, we used high-resolution respirometry to evaluate the effects of nitrite in an in vitro model I/R using hippocampal slices. We found that reoxygenation was accompanied by an increase in oxygen flux, a phenomenon that has been coined “oxidative burst”. The amplitude of this “oxidative burst” was decreased by nitrite in a concentration-dependent manner. These results support the notion that nitrite mediates a decrease in the hyper-reduction of the electron transport system during ischemia, decreasing the accelerated oxygen consumption that characterizes the reoxygenation phase of I/R that has been associated with an increase in oxidant production. Additionally, a pilot in vivo study in which animals received a nitrate-rich diet as a strategy to increase circulating and tissue levels of nitrite also revealed that the “oxidative burst” was decreased in the nitrate-treated animals. These results may provide mechanistic support to the observation of a protective effect of nitrite in situations of brain ischemia.

Oxidative stress is characterized by an excessive and prolonged formation of oxidants, causing an accumulating load of irreversible oxidative modifications of proteins, lipids, and nucleic acids that compromise cell integrity. This competes with the concept of redox regulation, combining the regulatory influence of nitric oxide (•NO), superoxide (O2•―), and their derivatives on redox-sensitive signaling pathways in the cell. The transition from redox regulation to oxidative stress is not only determined by the absolute amount of oxidants formed, but also by the respective intracellular site of formation, by the capacity of the defense machinery of the respective cell type, and by the ratio between •NO and O2•― that determines the nature of secondary radical species formed. Equimolar and concomitant fluxes of •NO and O2•―, for instance, favor the formation of the oxidant peroxynitrite making O2•― an antagonist of •NO as well as an inhibitor of prostacyclin synthesis, while an excess of •NO over O2•― supports the formation of nitrosating species. Secondary •NO-derived species hence not only define cellular targets affected but also the nature of posttranslational modifications. A profound knowledge of redox regulation and the conditions supporting its fluent transition into oxidative stress is hence of outermost importance in molecular cardiovascular medicine. The present overview therefore aims to determine the spectrum of •NO-derived reactive species and the cellular conditions characteristic for reversible modifications and their modulation of cellular targets in redox regulation. The second objective is to define preconditions in cardiovascular cells culminating in an expenditure of the cellular antioxidant system and an accumulation of irreversible modifications that compromise cellular functions to a point of no return.

Humans are complex holobionts in which many physiological functions are ensured by the gut microbiota. The communication between the microbiota and its human host relies on immune, neural, metabolic and endocrine pathways and the derailment of this interaction can lead to gastrointestinal and systemic diseases. Here, we propose a novel form of communication between the microbiota and the host, based on the production of redox species by gut bacteria and the activation of signaling cascades in host mucosa. The biological significance of such a pathway is further highlighted by the observation that these inter-kingdom interactions are modulated by dietary nitrate, the major precursor of nitrite and NO in vivo. We demonstrate that nitrate has a positive metabolic effect in a murine model of antibiotic-induced dysbiosis by regulating cecum morphology and body weight (p<0.05). In agreement with these observations, shallow shotgun sequencing analysis showed that nitrate modulates the metabolic function of bacteria involved in the metabolism of carbohydrates, likely aiding in food digestion and substrate delivery to the host. Furthermore, we observed that the exposure to antibiotics decreases the expression of tight junction proteins in the colon and that nitrate recovers the expression of both occludin (p<0.05) and claudin-5 (p<0.01). The activation of the Nrf2/ARE pathway was also investigated by the downstream expression of detoxifying enzymes including NQO1 and GCLM/GCLC. Here, dietary nitrate emerges as a pivot regulating microbiota-host interactions through redox pathways. Nitrate modulates the function of gut microbiota during dysbiosis by enhancing bacterial metabolic performance with positive effects on host body weight and prevents the loss of tight junction proteins likely reinforcing gut barrier integrity. Given that increased epithelial permeability may lead to leaky gut syndrome, triggering local and systemic disorders, this study has the potential to transform the way Redox Biology expands from the bench to patient's bedside.   

The physicochemical properties of nitric oxide (NO) as an intercellular messenger, in particular the way it conveys information via volume signaling, translate into advantages of communication in the brain. This becomes apparent when considering neurovascular coupling (NVC), the tightly temporal and spatial functional communication between active neurons and local blood microvessels. That the brain is energetically expensive given its mass and that increased neuronal activity in a region of the brain is associated with a local increase in blood flow (CBF) has been known since the XIX century. In turn, the association between CBF dysregulation and cognitive decline has been consistently established in older adults (brain aging, neurodegenerative diseases, type II DM) and lab rodent models but the neurobiological links are poorly understood. I will discuss the notion that neuronal-derived NO is the key mediator of NVC in the hippocampus and that impairment of NVC is an early and likely causative event leading to cognitive decline. The premise is that by rescuing the functionality of NVC then cognitive enhancement should be observed. This will be experimentally supported on basis of a diet-driven redox mechanism, involving the interaction of nitrite with ascorbate released from active neurons. Data suggest that an operational NVC, allocating energy resources according to neuronal activity, is a most fundamental biochemical process that underlines biological organization to support cognition.   

Supported by project 2022.05454.PTDC (https://doi.org/10.54499/2022.05454.PTDC).

The incidence of non-alcoholic fatty liver disease (NAFLD) is gradually increasing with the prevalence of obesity, which is the strongest risk factor for steatosis. Lipid droplet (LD) accumulation in hepatocytes is a hallmark of NAFLD. Seipin protein, which is LD related protein, resides in the endoplasmic reticulum membrane and a shortage of this protein leads to accumulation of abnormal LDs in adipose tissue. Although it has been shown that adipose-specific Seipin deficiency causes increased lipid accumulation in liver and muscle tissue following abnormal LD formation and loss of adipose tissue function, Seipin protein deficiency in liver tissue and its effect on lipid accumulation have not been investigated. Our study aimed to investigate the effect of Seipin deficiency on ER stress and lipophagy in cholesterol-accumulated mouse hepatocyte cells (AML12 cell line). In this direction cholesterol accumulation in mouse hepatocyte cells was established by administrating cholesterol-containing liposome and Seipin levels were reduced using siRNA transfection. Following liposome-cholesterol and siRNA administrations, lipophagy was determined by confocal microscopy, and mRNA levels of GRP78, GRP94, and ATF4 were examined by qRT-PCR. Our findings show that cholesterol-containing liposome administration in hepatocytes increases both Seipin protein and number of large LDs. However, Seipin silencing reduced the increase of cholesterol-mediated large LDs and GRP78 mRNA. Additionally, lysosome-LD colocalization increased only in cells treated with cholesterol-containing liposome, while the siRNA against Seipin did not lead to any significant difference. According to our results, we hypothesise that Seipin silencing in hepatocytes reduced cholesterol-mediated LD maturation as well as GRP78 levels, but not lipophagy.

Breast cancer is characterized by specific metabolic changes that support tumorigenesis, highlighting the emerging appreciation of cancer as a metabolic disease. These metabolic changes are simultaneous with redox reprogramming with nuclear factor erythroid 2-related factor 2 (Nrf2) representing their master integrator. Given that interscapular brown adipose tissue (IBAT) influences whole-body metabolism, our goal was to investigate the redox-metabolic crosstalk between the tumor and the host at the systemic level by exploring Nrf2-driven metabolic changes that occur in IBAT in the orthotopic model of breast cancer in wild-type (WT) and mice lacking functional Nrf2 (Nrf2KO). We analyzed the protein expression of key enzymes involved in glucose and lipid metabolism in control groups and at different points during tumor growth (10 mg, 50 mg, 100 mg, 200 mg, and 400 mg). In both WT and Nrf2KO mice, the results indicated a transient induction of hexokinase 2 expression during the early phase of tumor growth (<100 mg). Accordingly, pyruvate dehydrogenase expression followed the same profile. In Nrf2KO mice, a general decline in glyceraldehyde 3-phosphate dehydrogenase, phosphofructokinase-1, and glucose-6-phosphate dehydrogenase expression was detected during the late phase of tumor growth (>100 mg). Since no changes in WT mice occurred, these findings are considered Nrf2-dependent. Concomitantly, a decrease in protein expression of fatty acid synthase and acetyl-CoA carboxylase in Nrf2KO mice was observed. These observations correspond to decreased levels of 5'-AMP-activated protein kinase and hypoxia-inducible factor 1 during the late-phase (>100 mg) of tumor growth in Nrf2KO mice which suggests their involvement in transcriptional regulation. Our results revealed that IBAT metabolism responds to tumor growth and underscored that this communication is Nrf2-dependent giving implications for further understanding of breast cancer in the light of systemic metabolic disease.

This research was supported by the Science Fund of the Republic of Serbia, #7750238, Exploring new avenues in breast cancer research: Redox and metabolic reprogramming of cancer and associated adipose tissue - REFRAME.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that plays an essential role in the inflammatory response and various other biological effects such as activation of apoptosis and oxidative stress. Fructose-enriched diets have previously been associated with the development of low-grade inflammation leading to metabolic stress. The aim of the present study was to investigate the combined effects of deletion of the Mif gene and a 9-week 20% fructose-enriched diet on metabolic inflammation, apoptosis, and oxidative stress in the liver of wild-type (WT) and Mif knockout (MIF−/−) male C57Bl/6J mice. We analyzed liver histology and expression of pro-inflammatory genes: Tumor necrosis factor (TNF), interleukin 1β (IL-1β), and IL-6. Antioxidant activity was estimated by the protein levels of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD1), mitochondrial MnSOD (SOD2), glutathione reductase (GR) and glutathione peroxidase (GPX). The results showed that antioxidant protection was activated in the liver of MIF-deficient mice. Increased hepatic expression of the cytokines IL-6 and IL-1β was observed in the same animals. Histologic analysis confirmed the presence of apoptosis, inflammation, enlarged Kupffer cells, and regenerative changes, such as binucleated hepatocytes, anisonucleosis, and anisocytosis. In addition, confluent and focal necrosis was observed in the liver of MIF−/− mice, which was even more pronounced in the animals consuming fructose. In conclusion, MIF may play a protective role in metabolic stress, as inflammation, oxidative stress, apoptotic and necrotic changes occur in the liver in its absence.

The diverse uses of microalgae in ecological remediation, wastewater treatment, pharmaceutics, or food and biofuel production, have long kept these single-celled organisms in the spotlight. The focus of this study was on Chlamydomonas acidophila strain PM01, which thrives in acidic aquatic systems and is resistant to the presence of heavy metals in its environment. The redox metabolism of this microalga was assessed by its ability to reduce the EPR-active spin probe TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl), and compared to that of Chlorella sorokiniana strain CCAP 211/8K, a freshwater green microalga. The results showed that C. acidophila has a faster redox metabolic rate than C. sorokiniana, reducing 50% of TEMPO after 2.5, and 13 min, respectively. The addition of Mn²⁺ or Fe³⁺ to the culture medium of C. acidophila did not affect its reduction capacity, while it had a minor effect on C. sorokiniana. The faster rate in C. acidophila most likely represents the result of its adaptation to acidic environments. Namely, it has previously been suggested that acidophilic algae perform energy-demanding cellular processes in order to cope with the high pH gradient across the membrane. Moreover, the increased metabolic turnover requires an increased mitochondrial activity, resulting in a higher baseline production of superoxide and hydrogen-peroxide, subsequently compensated by an elevated baseline reduction capacity. Interestingly, the redox metabolic rate of C. sorokiniana was unaltered in suspensions that were kept in non-standard cultivation conditions (diurnal fluctuations of temperature and ambient lighting, absence of shaking) for five weeks. However, C. acidophila lost all of its reduction capacity in these conditions already after three days. These findings may be important when selecting the most appropriate microalgal strain for a specific application. Specifically, C. acidophila would likely be a good candidate for high-yield rapid production of endogenous products that are the result of its unique survival mechanism under extreme conditions.

Considering the crucial antioxidant role of glutathione (GSH) in cells, its assessment is useful for both healthy populations and in different diseases. It is usually measured either in erythrocytes or in plasma, while it is unknown whether the distribution of GSH between these compartments depends on a presence of a disease, thus affecting the results. Therefore, our aim was to investigate the relationship between GSH in plasma and erythrocytes of healthy and diseased subjects. The study included 60 participants, 25 healthy subjects, and 35 patients with different diseases (cancer, heart failure, kidney diseases, chronic fatigue, sarcoidosis, Lyme disease). GSH levels were determined in plasma and erythrocytes using spectrophotometric method with Ellman’s reagent. GSH plasma/erythrocytes ratio between two groups was compared by Mann-Whitney U test and the results are presented as median (interquartile range). The median value of plasma/erythrocytes ratio for healthy subjects was 3.79 (3.32-5.71), and for patients, it was 27.54 (1.53-54.76). This ratio was significantly higher in the group of patients compared to healthy participants (P=0.018). Our results indicate a redistribution of GSH from erythrocytes to plasma in the presence of different diseases. The fact that this preliminary study points out an association of health status with plasma/erythrocytes GSH ratio, regardless of the heterogeneity of a patient group, encourages further research in this direction.