This open access paper on mitochondrial function considers the mechanism of uncoupling in calorie restriction and in drugs that seek to emulate some of the benefits produced by calorie restriction, known as calorie restriction mimetics. Mitochondria generate energy stores for use in cells, but with greater uncoupling that effort creates heat instead. This is a part of the normal process of body temperature regulation in mammals. However, uncoupling also changes the output of reactive oxygen species (ROS) from the mitochondria, a feature observed in the methods shown to modestly slow aging in short-lived species. Mitochondrially generated ROS are both a signal that spurs greater cellular housekeeping and a source of damage, so either somewhat more or somewhat less than the usual output might be beneficial.
There are drugs known to reliably produce greater mitochondrial uncoupling, but there has little development of their use as therapeutics for aging, even now that the research community has more enthusiasm for the goal of slowing aging via pharmaceuticals. This is possibly because unbounded increases in uncoupling via drug administration are fairly dangerous: too much is potentially lethal due to raised body temperature and harmful effects on mitochondrial biochemistry. Since this lack of safety at the higher end is an inherent feature of uncoupling in mammals, it may well be the case that direct intervention in the uncoupling process will remain less desirable in comparison to the range of other potential approaches to modestly slow aging in humans. That said, the researchers here point out a family of self-limiting uncouplers that may not exhibit this problem; we shall see how it goes in the years ahead.
Caloric restriction (CR) is the best-studied and most reliable way to increase lifespan. CR affects most of experimental model organisms, from unicellular ones to mammals. Signaling cascades responsible for the effects of CR were studied in detail at the cellular level as well as at the levels of tissues and the whole organisms. Increasing levels of AMP and NAD+, which activates deacetylases, were shown to be the key factors initiating these cascades at the cellular level. One could expect that under the conditions of CR the cells attempt to save energy. Many cellular changes indeed make bioenergetics more economical: CR decreases the rate of protein biosynthesis and activates autophagy. It would be natural to presume that CR also raises the efficiency of mitochondrial energy production, i.e. that it increases the coupling of respiration and oxidative phosphorylation. However the opposite appears to take place.
It has been shown that mice under CR conditions accumulate UCP proteins (uncoupling proteins) in their muscle mitochondria. UCP proteins catalyze an electrogenic process of transporting the dissociated forms of free fatty acids from the inner to the outer layers of mitochondrial inner membrane. In the outer layer the free fatty acids are protonated, and then in the neutral form return to the inner layer. As this decreases the level of the transmembrane potential, the proteins of the UCP family act as natural uncouplers of respiration and oxidative phosphorylation. Indeed, during starvation there is simultaneous accumulation of UCP2 and UCP3, and a decrease a decrease in the efficiency of oxidative phosphorylation. What is the physiological role of uncoupling activation upon CR? On one hand, CR induces mitochondrial biogenesis and respiration. On the other hand, it has been shown that mitochondrial hyperpolarization can induce a strong increase in ROS (reactive oxygen species) generation. Probably, the increased expression of UCP is an "insurance" against the oxidative stress caused by mitochondrial hyperpolarization.
There is probably another advantage of using uncouplers as CR mimetics. As aforementioned, increase in NAD+ levels is one of the best-studied ways of geroprotection. There are many works showing that increasing NAD+ concentration via activation of its biosynthesis leads to lifespan increase in experimental animals. Importantly, in terms of lifespan increase in the sum of NAD+ and NADH concentrations is less relevant than the concentration of the oxidized form. At the same time, reduction of NAD+ to NADH takes place during cellular catabolic reactions (glycolysis and TCA cycle). Therefore, interfering with cell metabolism could be an efficient way of increasing NAD+ concentration. The addition of the uncoupler FCCP at low concentration has been shown to increase ATP level in neurons due to a compensatory response to a temporal depolarization. Earlier, it has been suggested that a slight decrease in the transmembrane potential can prevent the reaction of one-electron reduction of oxygen, which leads to ROS formation. At the same time, such a decrease may not affect the rate of ATP synthesis; thus, such uncoupling was called "mild". In other words, mild uncoupling is aimed at stimulating NAD+-dependent processes rather than at stimulation of AMPK.
What level of uncoupling is most suitable for the purposes of geroprotection? As mentioned, a small decrease in proton resistance of the strongly energized mitochondrial membranes can induce a significant decrease in ROS production and an increase in NAD+/NADH ratio without affecting ATP concentration. According to our line of reasoning, such level of uncoupling combined with AMPK activation is sufficient for efficient interference with the aging process. Theoretically, one could consider a higher level of uncoupling leading to a strong depolarization of the membranes and, as a consequence, a significant increase in ADP/ATP ratio. Apparently, such treatment could lead to a lethal deenergization of cells. Therefore, a relatively weak level of uncoupling seems to be preferential.
Which uncouplers should be used? Probably, the anionic compound dinitrophenol is the best-studied uncoupler in terms of its effects on mammalian physiology. In particular, it has been used on humans as a weight loss treatment. However, it was reported that its use was accompanied by a set of negative side effects. Recently, we reported uncoupling activity of a unique type of chemical compounds - lipophilic penetrating cations. Most of the studies on such compounds were performed on dodecyltriphenylphosphonium (C12TPP). A potential advantage of using such compounds is that their mitochondrial accumulation is proportional to the level of the transmembrane potential. For this reason, penetrating cations affect highly polarized mitochondria to greater extent than mitochondria with relatively low potential levels. In other words, they cause self-limiting (mild) uncoupling.