Bruno Chausse

Mitochondrial compartmentalization of redox processes

Knowledge of location and intracellular subcompartmentalization is essential for the understanding of redox processes, because oxidants, owing to their reactive nature, must be generated close to the molecules modified in both signaling and damaging processes. Here we discuss known redox characteristics of various mitochondrial microenvironments. Points covered are the locations of mitochondrial oxidant generation, characteristics of antioxidant systems in various mitochondrial compartments, and diffusion characteristics of oxidants in mitochondria. We also review techniques used to measure redox state in mitochondrial subcompartments, antioxidants targeted to mitochondrial subcompartments, and methodological concerns that must be addressed when using these tools.

Long-term intermittent feeding, but not caloric restriction, leads to redox imbalance, insulin receptor nitration, and glucose intolerance.

Calorie restriction is a dietary intervention known to improve redox state, glucose tolerance, and animal life span. Other interventions have been adopted as study models for caloric restriction, including nonsupplemented food restriction and intermittent, every-other-day feedings. We compared the short- and long-term effects of these interventions to ad libitum protocols and found that, although all restricted diets decrease body weight, intermittent feeding did not decrease intra-abdominal adiposity. Short-term calorie restriction and intermittent feeding presented similar results relative to glucose tolerance. Surprisingly, long-term intermittent feeding promoted glucose intolerance, without a loss in insulin receptor phosphorylation. Intermittent feeding substantially increased insulin receptor nitration in both intra-abdominal adipose tissue and muscle, a modification associated with receptor inactivation. All restricted diets enhanced nitric oxide synthase levels in the insulin-responsive adipose tissue and skeletal muscle. However, whereas calorie restriction improved tissue redox state, food restriction and intermittent feedings did not. In fact, long-term intermittent feeding resulted in largely enhanced tissue release of oxidants. Overall, our results show that restricted diets are significantly different in their effects on glucose tolerance and redox state when adopted long-term. Furthermore, we show that intermittent feeding can lead to oxidative insulin receptor inactivation and glucose intolerance.

Caloric restriction increases brain mitochondrial calcium retention capacity and protects against excitotoxicity

Caloric restriction (CR) protects against many cerebral pathological conditions that are associated with excitotoxic damage and calcium overload, although the mechanisms are still poorly understood. Here we show that CR strongly protects against excitotoxic insults in vitro and in vivo in a manner associated with significant changes in mitochondrial function. CR increases electron transport chain activity, enhances antioxidant defenses, and favors mitochondrial calcium retention capacity in the brain. These changes are accompanied by a decrease in cyclophilin D activity and acetylation and an increase in Sirt3 expression. This suggests that Sirt3‐mediated deacetylation and inhibition of cyclophilin D in CR promote the inhibition of mitochondrial permeability transition, resulting in enhanced mitochondrial calcium retention. Altogether, our results indicate that enhanced mitochondrial calcium retention capacity underlies the beneficial effects of CR against excitotoxic conditions. This protection may explain the many beneficial effects of CR in the aging brain.

Intermittent Fasting Results in Tissue-Specific Changes in Bioenergetics and Redox State

Intermittent fasting (IF) is a dietary intervention often used as an alternative to caloric restriction (CR) and characterized by 24 hour cycles alternating ad libitum feeding and fasting. Although the consequences of CR are well studied, the effects of IF on redox status are not. Here, we address the effects of IF on redox state markers in different tissues in order to uncover how changes in feeding frequency alter redox balance in rats. IF rats displayed lower body mass due to decreased energy conversion efficiency. Livers in IF rats presented increased mitochondrial respiratory capacity and enhanced levels of protein carbonyls. Surprisingly, IF animals also presented an increase in oxidative damage in the brain that was not related to changes in mitochondrial bioenergetics. Conversely, IF promoted a substantial protection against oxidative damage in the heart. No difference in mitochondrial bioenergetics or redox homeostasis was observed in skeletal muscles of IF animals. Overall, IF affects redox balance in a tissue-specific manner, leading to redox imbalance in the liver and brain and protection against oxidative damage in the heart.

Intermittent Fasting Induces Hypothalamic Modifications Resulting in Low Feeding Efficiency, Low Body Mass and Overeating

Intermittent fasting (IF) is an often-used intervention to decrease body mass. In male Sprague-Dawley rats, 24 hour cycles of IF result in light caloric restriction, reduced body mass gain, and significant decreases in the efficiency of energy conversion. Here, we study the metabolic effects of IF in order to uncover mechanisms involved in this lower energy conversion efficiency. After 3 weeks, IF animals displayed overeating during fed periods and lower body mass, accompanied by alterations in energy-related tissue mass. The lower efficiency of energy use was not due to uncoupling of muscle mitochondria. Enhanced lipid oxidation was observed during fasting days, whereas fed days were accompanied by higher metabolic rates. Furthermore, an increased expression of orexigenic neurotransmitters AGRP and NPY in the hypothalamus of IF animals was found, even on feeding days, which could explain the overeating pattern. Together, these effects provide a mechanistic explanation for the lower efficiency of energy conversion observed. Overall, we find that IF promotes changes in hypothalamic function that explain differences in body mass and caloric intake.

Diluted serum from calorie‐restricted animals promotes mitochondrial β‐cell adaptations and protect against glucolipotoxicity

β‐cells quickly adjust insulin secretion to oscillations in nutrients carried by the blood, acting as fuel sensors. However, most studies of β‐cell responses to nutrients do not discriminate between fuel levels and signaling components present in the circulation. Here we studied the effect of serum from calorie‐restricted rats versus serum from rats fed ad libitum, diluted tenfold in the medium, which did not contribute significantly to the pool of nutrients, on β‐cell mitochondrial function and dynamics under regular and high‐nutrient culture conditions. Insulin secreting beta‐cell derived line (INS1) cells incubated with serum from calorie‐restricted rats (CR serum) showed higher levels of peroxisome proliferator‐activated receptor gamma coactivator 1‐α (PGC‐1α) and active nitric oxide synthase. The expression of mitofusin‐2 (Mfn‐2) and optic atrophy 1 (OPA‐1), proteins involved in mitochondrial fusion, was increased, while the levels of the mitochondrial fission mediator dynamin related protein 1 (DRP‐1) were reduced. Consistent with changes in mitochondrial dynamics protein levels, CR serum treatment increased mitochondrial fusion rates, as well as their length and connectivity. These changes in mitochondrial morphology were associated with prolonged glucose‐stimulated insulin secretion and mitochondrial respiration. When combining CR serum and high levels of glucose and palmitate (20 and 0.4 mm, respectively), an in vitro model of type II diabetes, we observed that signaling promoted by CR serum was enough to overcome glucolipotoxicity, as indicated by CR‐mediated prevention of mitochondrial fusion arrest and reduced respiratory function in INS1 cells under glucolipotoxicity. Overall, our results provide evidence that non‐nutrient factors in serum have a major impact on β‐cell mitochondrial adaptations to changes in metabolism.

Bioenergetic profiling in the skin

Background The skin is a large organ which presents important thermoregulatory and metabolic functions (1,2) and should thus be a focus of studies involving energy metabolism. Nevertheless, bioenergetic studies in epithelia-containing organs such as the skin are rare (3,4), probably due to difficulties in organelle isolation (5) or in situ studies in these tissues, added to the lack of published protocols. Furthermore, the skin is subdivided into two tissues: the dermis and the epidermis, as is typical in the structure of composite organs with an epithelial tissue on top of a mesenchymal layer. Results obtained with whole-organ homogenates in composite tissues such as the skin tend to be the average of the individual responses of the different types of cells resident in the tissue (6). It is thus also important to establish techniques that allow for measurements of bioenergetic characteristics in different tissues of the skin. Questions addressed We established techniques to study skin mitochondrial bioenergetics in isolated organelles and in situ in different cell types, producing a bioenergetic profile of this tissue. These methods can also be easily adapted to human skin with evident clinical relevance (S1). Experimental design Techniques used are described in detail in the Supporting information. Mitochondrial isolation and oxygen consumption Mitochondria were isolated from mouse skin samples using an adaptation of standard differential centrifugation methods. Oxygen concentrations and consumption rates (OCR) were measured using Oroboros high-resolution respirometry (3). Cell isolation, culture and oxygen consumption The dermis and epidermis of mouse back skins were separated by scraping, and the two tissues were cultured separately. The epidermal fraction consisted mainly of keratinocytes (89 2.2%) and the dermal fraction of fibroblasts (88 1.6%, Table S1). OCR and extracellular acidification rates (ECAR) were determined using an XF24 extracellular flux analyser (Seahorse Bioscience, North Billerica, USA). Results By adding trypsin digestion and fur-filtering steps to standard differential centrifugation protocols, we were able to isolate highly functional mitochondria from murine whole-skin samples. Figure 1 shows a typical oxygen tension trace (Panel a) and quantified ADPmaximized OCR (Panel b) supported by different respiratory substrates (NADH-linked pyruvate and malate, Complex II electron donor succinate and Complex IV electron donor TMPD). The relative contribution of each respiratory complex was uncovered using specific inhibitors: rotenone for Complex I and antimycin A for Complex III. Skin mitochondria respire well with either pyruvate plus malate or succinate as substrates. To assess the quality of the preparations, we measured respiration in the presence and absence of ATP synthesis (7). Figure 1c and d indicates that skin mitochondria are highly coupled, presenting a large increase in OCR when ADP is added, in a manner inhibited by oligomycin, an ATP synthase inhibitor. The respiratory control ratio was on average 4.39, a value similar to that obtained in preparations from mesenchymal tissues (7) and indicating that integrity was maintained. The addition of the protonophore CCCP stimulated the oligomycin-inhibited OCR, further indicating that these mitochondria were fully coupled. Overall, we find that the method described provides adequate quantities of high-quality mitochondria. 0 3 6 9 12 150 200 250 300 Pyruvate + Malate

Cell culture models of fatty acid overload: Problems and solutions

High plasma levels of fatty acids occur in a variety of metabolic diseases. Cellular effects of fatty acid overload resulting in negative cellular responses (lipotoxicity) are often studied in vitro, in an attempt to understand mechanisms involved in these diseases. Fatty acids are poorly soluble, and thus usually studied when complexed to albumins such as bovine serum albumin (BSA). The conjugation of fatty acids to albumin requires care pertaining to preparation of the solutions, effective free fatty acid concentrations, use of different fatty acid species, types of BSA, appropriate controls and ensuring cellular fatty acid uptake. This review discusses lipotoxicity models, the potential problems encountered when using these cellular models, as well as practical solutions for difficulties encountered.