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Drug discovery (SR9009): Time in a bottle
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Download the PDF here
Nature
02 May 2012
A biological clock synchronizes animal behaviour and physiology with Earth's 24-hour rotation. Drugs targeting the clock's 'gears' show promise for treating obesity and other metabolic disorders. See Article p.62
When spring approaches, many countries set their clocks forward by one hour. The following morning, we are reminded of the strong pull exerted by our own internal clock as we are forced to wake up one hour earlier than usual. Such minor disruption of sleep patterns can lead to fatigue and is associated with a rise in the incidence of certain heart disorders1. These undesirable effects occur because our internal clock - which tracks the circadian (day-night) cycle - controls not only our sleep patterns but also many physiological processes that anticipate the rhythmic environmental changes tied to the Sun's rising and setting. In this issue, Solt et al.2 (page 62) and Cho et al.3 (page 123) illuminate the molecular mechanisms by which the circadian clock regulates metabolism in mice, and provide evidence to suggest that drugs targeting clock components may offer treatment for disorders such as obesity and diabetes.
In the late 1990s, the finding4 of biological rhythms in cultured fibroblast cells indicated a broad role for the circadian clock in cell physiology. Subsequently, circadian oscillations were observed5 in the expression of at least 10% of the genome in mouse tissues. At the molecular level, the circadian clock consists of a feedback loop that involves activator and repressor proteins, and repeats itself every 24 hours. Activators induce the expression of repressors, whereas repressors inhibit activators' expression.
One of these repressors is REV-ERB-α, a member of the nuclear-receptor family of proteins6. In addition to controlling the expression of activators' genes7, REV-ERB-α modulates the production of lipids and bile acids in the liver8, 9, and the formation of fat cells10. Whereas the activity of most nuclear receptors is induced on binding to specific steroid hormones, REV-ERB-α has been shown11, 12 to bind instead to haem (an oxygen-binding molecule) and, in turn, to regulate haem synthesis13. These findings have increased interest in the development of synthetic compounds that, by binding to REV-ERB-α, could modulate the protein's function.
Notwithstanding these advances, the part played by REV-ERB-α in the circadian clock has remained enigmatic because mice lacking this protein show relatively minor defects in their behavioural rhythms7. A possible explanation for this is that a closely related protein, REV-ERB-ß, can compensate for REV-ERB-α deficiency, as suggested by studies14 of cultured cells.
To understand the circadian functions of the two REV-ERB proteins, Cho et al.3 identified the genomic regions that these repressors occupy in the mouse liver.
This analysis revealed that both proteins bind to regulatory regions of genes encoding not only numerous core components of the clock but also proteins involved in various metabolic pathways. Therefore, REV-ERBs probably control circadian oscillations through effects beyond modulation of the clock activators. Whether the action of REV-ERBs on clock genes is crucial for the oscillations in cellular activity in other organs - such as the brain - requires additional study.
Mice deficient in REV-ERB-α have been shown7 to have an increased mortality. So, to carry out experiments with mice lacking both REV-ERB proteins, Cho et al. used a genetic-engineering technique known as Cre/lox recombination to generate a mouse strain in which the simultaneous deletion of both genes could be experimentally induced in adulthood. The authors monitored these double-mutant mice running in wheels as a test for circadian dysfunction, and found that the animals' running rhythms had a markedly shortened period length when compared with those of control animals. Moreover, the double mutants displayed an altered response to light.
The researchers compared several metabolic parameters of the double-mutant mice with those of control littermates. The mutant mice had elevated blood levels of triglyceride lipids and of glucose, decreased levels of free fatty acids and a lower respiratory exchange ratio (the relative amount of exhaled carbon dioxide and inhaled oxygen). These metabolic alterations are consistent with an increased generation of energy from fat in the mutant mice.
Cho and colleagues' work3 provides additional evidence for REV-ERBs as central elements of the circadian clock, and demonstrates that these proteins participate in the control of liver metabolism. To gain further insight into the functions of REV-ERBs, additional analyses of oxidative metabolism - the process by which cells obtain energy from the oxidation of organic compounds - and exercise tolerance in the mutant mice would be needed. For example, metabolic indicators could be monitored across time and under dynamic conditions, such as during a high-fat diet.
Enter Solt and colleagues2. Using a high-throughput screen against the entire family of nuclear hormone receptors in cultured human cells, the authors identified a group of related molecules that selectively activated REV-ERB-α and REV-ERB-ß. Two of these compounds were suitable for studies in mice, and were investigated further.
The researchers found that the compounds reduced the amplitude of the oscillations in clock-gene expression in cultured cells. And, when injected into mice, the drugs repressed the expression of clock genes. Indeed, the treated mice displayed altered wheel-running rhythms in constant darkness, but not under standard light-dark conditions (12 hours light, 12 hours dark). The cause of reduced drug activity under light-dark conditions on the animals' behaviour requires further investigation but may reflect a direct response to light that bypasses the clock mechanisms - an effect known as 'masking'. The authors carried out further studies in cultured cells that support the idea that the drugs' effects in mice are due to activation of REV-ERBs and not to modulation of other proteins.
Solt et al. report that, in addition to the actions on the circadian clock, the compounds protected the animals from certain metabolic disorders associated with obesity and high-fat feeding. The treated mice showed resistance to diet-induced obesity and an increased consumption of oxygen, as well as a reduced food intake during the light period when they are usually sleeping. Moreover, the animals had an altered profile of gene expression in the liver, fat and muscle. In particular, changes in the expression of enzymes involved in the metabolism and transport of fatty acids point towards enhanced oxidative metabolism and reduced lipid storage. The drug treatment also ameliorated metabolic alterations in genetically obese mice that lacked the hormone leptin.
Overall, the results reported by Cho et al.3 and Solt et al.2 re-emphasize the tight coupling of the circadian clock with metabolism, and the special role of REV-ERBs as a nodal point in this relationship. They also suggest that these nuclear receptors may repress the expression of more clock components than previously thought.
Furthermore, the studies raise the possibility of 'putting time in a bottle' - the development of drugs to manipulate biological clocks - for the treatment of metabolic disorders. Admittedly, such an effort entails a chicken-and-egg riddle: any compounds targeting REV-ERBs' activities may affect metabolic parameters either directly, by modulating the expression of metabolic targets, or indirectly, through effects on the clock. Moreover, as REV-ERB proteins are produced in an oscillatory manner, the actions of any drug would be limited to the window of REV-ERB expression.
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