Role of peripheral clock genes in the circadian regulation of the hunger hormone ghrelin.

The daily rotation of the earth around its axis creates a recurring succession of day and night with a period of 24 hours. As a consequence, life on earth is subjected to cyclical and therefore predictable changes in environmental conditions. In order to anticipate these daily events and fine-tune physiology to the varying demands of activity and rest, virtually all organisms have developed an internal timekeeping system. Circadian rhythms, derived from the Latin circa diem (“about a day”), are generated and sustained by a network of genetically encoded molecular clocks that impose this 24-h rhythmicity on downstream metabolic and physiological processes. However, our 24/7-society requires an increasing number of people to challenge their circadian system because of rotating shift work and chronic jet lag, thereby favoring the development of metabolic diseases such as obesity and type 2 diabetes.
To ensure proper timing, the circadian network requires external information to remain synchronized with (“entrained to”) the outside world. In general, photic information from the light/dark-cycle will entrain the hypothalamic central clock, which further conveys this 24-h rhythmicity to the periphery via hormonal and neuronal signals. Besides the light/dark-cycle, food also serves as an important synchronizer of peripheral clocks, even in mice with lesions in the master clock. Therefore, it has been postulated that the circadian rhythm of feeding is regulated by a separate network of food-entrainable oscillators (FEOs). When food availability is shifted to unusual times of day, the peripheral clocks will re-entrain to the new feeding rhythm and become uncoupled from the central clock to increase survival chances. As peripheral clocks are indispensable for the local control of physiology and metabolism, research has focused on finding the anatomical location and output signals of these FEOs. Recently, the ghrelin-secreting cells of the stomach were postulated to serve as one of the FEOs. Since ghrelin is a pleiotropic hormone which exerts numerous metabolic effects after its activation by the enzyme ghrelin O-acyltransferase (GOAT), including stimulation of food intake and body weight, this gut peptide represents the ideal candidate to integrate metabolic information into the circadian system and vice versa. However, it remains to be elucidated how circadian rhythmicity of ghrelin synthesis and/or secretion is controlled by the gastric molecular clock. Moreover, it is unclear whether ghrelin signaling plays a role in the triggering of physiological adaptations to alterations in the circadian rhythm of feeding.
Given that further understanding of the interaction between ghrelin and the circadian system might offer interesting prospects to prevent or counteract the harmful impact of chronodisruption on health, the aim of this PhD thesis was twofold. First, we aimed to explore the role of the clock gene Bmal1 and the gastric ghrelin-secreting cell as FEO in the circadian rhythmicity of the ghrelin-GOAT system. Second, we wanted to unravel the role of ghrelin and Bmal1 in the physiological adaptations induced by shifting the circadian rhythm of feeding.

In the first part of this PhD thesis, we demonstrated that Bmal1 is a pivotal regulator of the ghrelin‑GOAT system in mice. In contrast to their wild-type (WT) littermates, ad libitum-fed Bmal1 knockout (Bmal1-KO) mice lacked circadian rhythmicity in their feeding pattern and did not display 24-h fluctuations in their plasma ghrelin levels and gastric ghrelin and GOAT mRNA expression levels. In addition, lower mRNA expression levels of several components of the ghrelin signaling pathway were observed. These findings indicate that Bmal1 is required for optimal functioning of the ghrelin‑GOAT system and hence metabolism. In addition, using a murine MGN3-1 ghrelinoma cell line, we showed that in the absence of the master clock, food-related cues can entrain the molecular clock in the gastric ghrelin-secreting cell to regulate the rhythmic secretion of ghrelin. Surprisingly, transfection of the ghrelinoma cells with siRNA specific for Bmal1 did not alter the secretory response of the ghrelin-secreting cells towards feeding or fasting cues. Knockdown of Bmal1 might have been counteracted by other components of the circadian system, thereby ensuring the resilience of the molecular clock. Moreover, we observed diverging responses in octanoyl and total ghrelin release towards the feeding cue peptone. Together with the rhythmic expression of GOAT but not of ghrelin mRNA in the ghrelinoma cells, these findings indicate that the ghrelin cell as FEO not only regulates the circadian rhythmicity of ghrelin, but perhaps more importantly that of GOAT.

Night shift workers often present with obesity and related metabolic diseases that might be evoked by internal desynchronization, because their central clock stays entrained by photic information, whereas their peripheral clocks realign to the altered rhythm of food availability. A comparable desynchronization occurs during restricted feeding (RF), a paradigm in which nocturnal mice have ad libitum access to food, but only during the daytime and within a limited time window (for instance of 4 hours), for several weeks. Although previously postulated, it remains to be elucidated whether ghrelin serves as an output signal of the FEO that triggers physiological adaptations to alterations in the circadian rhythm of feeding. Therefore, the second part of this PhD thesis aimed to investigate the role of ghrelin in the RF-induced alterations in food intake, body weight and gastric emptying by comparing the effects in WT and ghrelin receptor knockout (GHSR-KO) mice.
Two weeks of RF induced a food-anticipatory increase in plasma ghrelin levels of both WT and GHSR‑KO mice. Over the course of the schedule, body weight restoration was facilitated by ghrelin. RF triggered contractility changes resulting in an acceleration of gastric emptying, which occurred independently from ghrelin signaling. Although ghrelin is known to exert anti-inflammatory effects, RF altered cytokine mRNA expression in both WT and GHSR-KO mice to a similar extent.
As these results indicated that the role of ghrelin in the physiological adaptations to RF was limited to the restoration of body weight loss, our focus shifted to the circadian system itself. Subsequently, we assessed whether the clock gene Bmal1 might serve as the key driver behind the adaptations by studying the effects of RF in Bmal1-KO mice. In accordance with the findings of the first part of this PhD thesis that indicate that Bmal1 plays a pivotal role in the circadian regulation of food intake and ghrelin signaling, Bmal1-KO mice failed to adapt to the RF schedule and died within 4 days, due to a persisting decrease in food intake and body weight. Therefore, the RF schedule was adjusted in several ways: food availability was decreased gradually and normal chow was replaced by a high-fat diet to increase the caloric meal content. RF with a high-fat diet did not induce a food-anticipatory increase in plasma ghrelin levels. In contrast to WT mice, Bmal1-KO mice were protected from increased body weight and fat pad mass during RF with a high-fat diet. In addition, the high-fat diet modulated the cytokine mRNA expression profile and resulted in a heightened proinflammatory state in the stomach right before food becomes available. The type of inflammatory cells infiltrating the stomach was modulated by Bmal1, which specifically promoted the infiltration of neutrophils and upregulated IL-1α expression. RF with a high-fat diet also resulted in a thickening of the gastric smooth muscle layers, which was counteracted by Bmal1. Our findings suggest that this thickening, whether or not in combination with the proinflammatory state, elicits the observed gastric smooth muscle hyperexcitability during RF.
In conclusion, we showed that disruption of the circadian rhythm of feeding, elicited by RF, induces a variety of diet-dependent metabolic, immune and gastrointestinal alterations. Moreover, we demonstrated that ghrelin and Bmal1 regulate the extent of body weight restoration during RF, whereas Bmal1 controls the type of inflammatory infiltrate and contractility changes in the stomach. Together, these findings emphasize the importance of the timing of food intake and partially explain the high incidence of obesity, metabolic and immune-related gastrointestinal disorders in shift workers or frequent time zone travelers.

Collectively, the results of this PhD thesis highlight the crucial role of the circadian system in the ghrelin-GOAT system and the overall maintenance of energy balance, during periods of both normal and shifted food availability. Moreover, it confirms that the ever increasing incidence of metabolic disorders is in fact related to disruption or desynchronization of the different circadian clocks, which is typical of our modern-day society. Nevertheless, the complex interactions of the circadian system with other organ systems make further research indispensable in order to fully understand the impact of altering the body clock in a lifestyle-associated or pharmacological way.