Adipose tissue mitchondrial function is modulated by antioxidants

Abstract

Antioxidants are widely used as reactive oxygen species (ROS) scavenging agents in experimental research and in conditions where oxidative stress plays a primary role. However, the effect of antioxidant supplementation on white and brown adipose tissue functionality is understudied, and the role of ROS and/or antioxidant treatment during adipose tissue browning, a process in which the adipocytes’ mitochondrial density and activity increase, is largely unknown. In paper I, by using antioxidants and ROS-sensitive fluorescent probes in cultured β3-AR-stimulated adipocytes, we observed that 24-48-hour antioxidant treatment increases the mitochondrial ROS production associated with reduced respiration and increased glycolysis. Moreover, treatment of mice with the antioxidant N-acetylcysteine (NAC) blunted the β3-AR agonist-induced browning response of white adipose tissue and reduced the mitochondrial activity in brown adipose tissue even in the absence of β3-AR stimulation. Previous studies have shown positive effects of prolonged NAC treatment on whole-body metabolism in mice. In light of these seemingly contradictory results, we hypothesize that chronic antioxidant exposure, in a dose-dependent manner, can lead to so-called mitohormesis. Indeed, in paper II, by treating mice with a set of different NAC doses across a defined time course, we found that prolonged supplementation with a high dose of NAC leads to increased mitochondrial function of white adipose tissue, reduced fat mass and improved insulin sensitivity. In summary, this thesis demonstrates that the adipose tissue response to antioxidant treatment in mice is biphasic and tightly connected to the adipose tissue type, the dosage and the treatment duration. This thesis also provides an alternative explanation for previously reported controversial findings where antioxidants (such as NAC) have exerted deleterious effects on health. Finally, the results of this thesis provide new insights into the appropriate design of antioxidant treatment studies: optimizing treatment dosage and duration may be the key to achieve success with antioxidant therapy.Based on previous research, we hypothesized that reactive oxygen species (ROS)/redox signaling plays a role in β3-AR agonist-induced browning. To test this hypothesis, we analyzed the effect of antioxidant treatment on β3-AR agonist-induced browning in cultured adipocytes. Using ROS-sensitive fluorescent probes to measure changes in total and mitochondrial ROS production in combination with Seahorse technology-based oxygen consumption measurements, we made the surprising observation that antioxidant treatment has a negative impact on adipocyte mitochondrial function even in absence of β3-AR-stimulation. This effect associated with increased mitochondrial ROS production. While similar pro-oxidant effects of antioxidants - sometimes referred to as reductive stress, where electrons are donated to oxygen leading to increased ROS-production - have been reported in a few other studies, it’s an understudied and, in our opinion, an underappreciated phenomenon. In fact, antioxidants are routinely used as a therapeutic or experimental tool to neutralize ROS and one may not necessarily detect potential pro-oxidant effects within the mitochondria unless such impact is directly measured. Therefore, we decided to expand our study and examine whether this deleterious effect of antioxidants also occurs in vivo using wild type mice. In line with our findings in vitro, treatment with the antioxidant N-acetylcystein blunts the β3-AR agonist browning response of white adipose tissue as well as reduces the mitochondrial activity in brown adipose tissue even in the absence of β3-AR stimulation, as it is observed by CARS microscopy in explanted tissue. Other studies have however shown positive effects of prolonged N-acetylcystein treatment on adipose tissue functionality and whole-body metabolism in mice. While such findings may seem contradictory to ours, we argued that prolonged N-acetylcystein exposure may lead to a so-called mitohormesis effect where the initial increase in mitochondrial ROS production upregulates the endogenous antioxidant system leading to positive effects on mitochondrial function in the long run. Indeed, we found that adipose tissue levels of mitochondrial antioxidant proteins in N-acetylcystein treated mice are increased. Moreover, prolonged treatment with a high dose of N-acetylcystein (in contrast to shorter or lower dose treatment regimens) is associated with increased mitochondrial function of white and brown adipose tissue and improved insulin sensitivity as judged by respectively, increased mRNA and protein expression of browning markers and reduced fasting insulin levels. The therapeutic effects of antioxidants (such as N-acetylcystein) have been studied in a broad range of health conditions including obesity, neurodegenerative diseases and cancer. The results of these studies are inconclusive. We believe that our data open up the possibility that positive and negative effects of antioxidant treatment on disease progression may not chiefly depend on ROS neutralization, but can be due to reductive stress that in a dose- and time-dependent manner may lead to an adaptive upregulation of the endogenous antioxidant system.