Molecular and neural implications of the body clock in food addiction: illuminating the avenue against obesity – ADDiCLOCK

We used Rev-erb-alpha mutant mice and their wild-type littermates. Moreover, we used transgenic animals (mice) bearing a DNA construct in which the PER2 protein promoter is fused to a cDNA encoding firefly luciferase reporter, which could be used as a circadian reporter gene. Real-time luminescence offers the opportunity to measure the expression of circadian genes and proteins. One advantage of this technique is that measurement from individual tissue samples reduces the number of experimental animals used and the inter sample variability. Circadian rhythms (phase, period, amplitude) can be measured from whole animal and from cultured tissues or specific organs. Bioluminescence tissue culture recordings will be continuously monitored with a photomultiplier tube (PMT) detector system (LumiCycle, Actimetrics). Mapping of the circadian pattern of clock genes expression will be determined in the brain of animals by in situ hybridization or rtPCR. Protein expression will be evaluated by immunohistochemistry or western blot.
Here we use the feeding paradigm of free choice to high-fat and high-sucrose diets (snacking behavior; fcHFHS, la Fleur et al., 2010). We will measure food-addicted behaviors (e.g. overeating, sensitization-like behavior) as addiction signs in our models.
The daily rhythms of locomotor activity, wheel-running behavior and body temperature will be studied by telemetry (Actimetrcis, CAMS). Moreover, behavioral studies related to food-reward and food-addiction (e.g. conditioned place preference, sensitization, and diet preferences) will be used as well.

Using Rev-erb-alpha mutant mice, we observed an increased expression of the neuropeptide orexin in the hypothalamus. Orexins are produced and released in the brain by neurons in the lateral hypothalamus, during the animal's active phase (phase during which animals eat). We went further by demonstrating a molecular link between the circadian clock and the orexinergic system. Orexin expression is down- regulated by Rev-erb-alpha. Thus, mutant mice show an increase in the mRNA and protein expression of Orexin. This is correlated, physiologically and behaviorally, with an increased preference for palatable foods such as sucrose or chocolate. Rev-erb-alpha mutant mice consume caloric foods in greater quantities than wild type mice, and then they become obese faster. Moreover, using addiction behavioral tests, we observed that Rev-erb-alpha mutant mice have a higher motivation for sweet rewards, and they over consume without moderation. How to explain that the disruption of the circadian clock, by disabling Rev-erb-alpha, may have such consequences? We demonstrate that this effect is the result of increased orexinergic activity in the lateral hypothalamus, since the compulsive feeding behavior for tasty foods returns to normal following an injection of an orexin antagonist. Our data highlight an implication of the circadian clockwork in modulating food-reward behaviors with an important impact for the central regulation of overeating.

One of the current perspectives is to study the effects of a perturbation of the circadian system in compulsive feeding behavior. Therefore, animals exposed to chronic changes of the light-dark cycle (Social jet-lag and a shift-work models) will be exposed to a compulsive food taking model, animals have a free choice between standard chow and a palatable cafeteria diet (lard, chocolate), or the snacking model of fcHFHS. Using the PER2 Luciferase transgenic mice, brains will be monitored for bioluminescence activity in vivo. Currently, in the group we develop a method to measure circadian rhythms in freely behaving animals using optic fibers implanted into specific brain substrates. Moreover, a bioluminescence microscopy (Luminoview) will be used for long-term recording of the expression of clock genes in imaged brain slices. This allows us to analyze the gross expression of clock genes in the whole culture, as well as view each individual cell.

Two articles were published (Addiction Biology; Frontiers in Psychiatry), and another manuscript was submitted to Scientific Reports. These three studies largely cover the first part of the project, mainly we showed the involvement of a clock gene (Rev-erb-alpha) as a modulator of brain systems involved in food intake and addiction (Addiction Biology); and also the aberrant effects of light exposure at night on the circadian system and feeding behavior (Scientific Reports).

In industrialized countries, we observe a dramatic increase in obesity probably due to excessive or compulsive eating. Beyond metabolic need, palatability and reward are thought to be major determinants for food intake of particular palatable foods. Overeating of highly palatable foods can alter brain function in specific regions of the reward system (e.g. the mesolimbic dopamine pathway) in a way similar to drugs of abuse such as cocaine. The neuronal and molecular mechanisms implicated in “food addiction” are unclear. In the brain, food intake is regulated by two compensatory mechanisms, a homeostatic and a hedonic mechanism. Importantly, food intake is also modulated by the circadian clock in the brain; the hypothalamic suprachiasmatic nuclei (SCN). Interestingly, the SCN clock, and its molecular machinery (clock gene expression), are implicated in the regulation of drug intake, and then could also regulate food compulsive behaviors. Nevertheless, the circadian neural and molecular mechanisms implicated in compulsive food-taking behavior are still poorly explored. Circadian activity of the SCN, which is mainly synchronized to the light-dark cycle, is molecularly built up by interlocked feedback loops encoded by a series of clock genes. The nuclear receptor Rev-erb-alpha is a key gene for the body clock and plays a crucial regulatory role as a molecular link between circadian rhythms, metabolism and behavior. Thus, Rev-erb-alpha seems appears as a good candidate for the control of metabolism and food-addictive behaviors. So far, the role of Rev-erb-alpha in the brain is still poorly understood and it may be relevant for the understanding on the circadian system implications in food-addicted disorders, and for the proposition of next generation treatments against obesity. Therefore, goals in this project are (1) to evaluate whether overconsumption of palatable foods affects the expression of the clock gene Rev-erb-alpha in brain structures regulating reward and motivation; (2) to characterize whether a specific mutation of the Rev-erb-alpha may induce a compulsive food intake and to understand the neuronal and molecular mechanisms underlying. (3) To evaluate whether an alteration of the circadian system, by abrupt shifts in the light/dark (LD) cycle (jet-lag or shift-work models) induce a preference and an overeating of highly palatable foods. Central mechanisms underlying these alterations will be evaluated. (4) To study the effects of light (important synchronizer of the circadian system) on the regulation of palatable food intake and the neuronal and molecular mechanisms implicated. This study will help to understand, the role of the circadian system in the regulation of the hedonic drive of feeding behavior implicated in the development of obesity, and to propose preventive treatments (light therapy).