Obesity and related metabolic disorders such as type 2 diabetes are a major health issue of our modern society. The brain has been identified to play an essential role in the pathogenesis of these diseases. Disruptions of the neuroendocrine system, such as the development of hypothalamic leptin resistance, are strongly correlated with the manifestation of diet-induced obesity (DIO). To date, the molecular mechanisms underlying these metabolic derangements are incompletely understood. Over the last decade, a close connection of energy metabolism and the circadian clock has been established, but the link between DIO and disruptions of physiological rhythms still needs further investigation. Therefore, the aim of this thesis was to gain new insights into neuroendocrine mechanisms that lead to the development of leptin resistance and the role of physiological rhythms in the disruption of energy metabolism.
In this study, we investigated the implication of the adipocyte-derived hormone adiponectin in neuroendocrine control of energy metabolism. We detected expression of all investigated genes involved in the adiponectin signalling pathway in the hypothalamus of mice. Expression levels of adiponectin were reduced during states of food deprivation, potentially presenting a regulatory mechanism to counteract the anorexigenic traits that had been previously described for central adiponectin signalling and that were confirmed by us in this study, in order to prevent further reduction in body weight. In both fasted control mice as well as DIO mice, gene expression of the adiponectin receptor AdipoR1 was elevated, suggesting multiple regulatory mechanisms to maintain sufficient adiponectin signal transduction. The upregulation of AdipoR1 during DIO might be an attempt to support the beneficial effects of the hormone on metabolic health that have been reported for peripheral adiponectin. In line with this, we demonstrated that adiponectin holds insulin-sensitising, blood glucose-lowering and anti-inflammatory properties in control as well as DIO mice and that these effects are mediated via central signal transduction.
We furthermore investigated the role of the WNT/β-catenin pathway in the neuroendocrine control of energy metabolism. Here, we found gene expression of members of the WNT pathway on all regulatory levels (ligands, intracellular pathway enzymes, target genes) in the hypothalamus of adult Djungarian hamsters, Phodopus sungorus, a seasonal rodent that exhibits profound annual changes in body weight and leptin sensitivity. Expression of all ligands as well as target genes was upregulated in hamsters acclimated to long day (LD) relative to short day (SD) conditions. Confirming our results from these transcriptional studies, we furthermore found increased phosphorylation of the WNT pathway co-receptor LRP-6, demonstrating elevated activation of canonical WNT signalling, in LD hamsters. These findings provide strong evidence for increased WNT signalling during LD compared with SD photoperiod. We found a 24-hour rhythm in the hypothalamic expression of WNT target genes, with decreasing levels during the light and increasing levels during the dark phase in both LD and SD hamsters. Moreover, leptin administration led to a further increase in LRP-6 activation in hamsters from both photoperiods. Taken together, we demonstrate a novel integration site for the leptin signal in the hypothalamus, potentially linking the WNT pathway to body weight regulation. Furthermore, our results suggest an important role of canonical WNT signalling in the seasonal as well as daily neuroendocrine control of energy metabolism in Djungarian hamsters.
By examining whether hypothalamic leptin signalling and whole body metabolism are modulated by a daily rhythm, we detected a 24-hour rhythm of STAT3 phosphorylation, a marker for activated leptin signalling on a molecular level, in the hypothalamus of wild-type mice. Both basal as well as leptin-induced leptin sensitivity were highest at the end of the dark (active) phase and lowest at the end of the light (inactive) phase. Furthermore, we found that leptin sensitivity on a behavioural level followed the same rhythm, with mice showing a greater response to exogenous leptin at the end of the dark phase at Zeitgeber time (ZT) 0 compared with the end of the light phase at ZT12. Throughout the 24-hour cycle, mice displayed a robust rhythm in food intake, locomotor activity as well as oxygen consumption and energy expenditure, with reduced whole body metabolism during their inactive and increased metabolic rate during their active phase. In DIO mice that were subjected to high-fat diet (HFD) feeding, we found a disruption of the 24-hour rhythmic regulation of leptin pathway activation on a molecular level for both basal and leptin-induced leptin sensitivity. Intriguingly, we demonstrated that this hypothalamic leptin resistance is a temporary phenomenon that persists only at specific times during the day. Responsiveness to leptin was deteriorated during the second part of the dark and the first half of the light phase (ZT21 – ZT6), but identical to mice fed low-fat diet (LFD) at all other times on both the molecular and behavioural level. Furthermore, DIO mice displayed a disruption of the daily rhythms in food intake, locomotor activity, oxygen consumption and energy expenditure. We found that the daily caloric overconsumption observed in mice fed HFD was restricted to the phase when DIO mice were leptin resistant relative to mice fed LFD. In conclusion, these findings provide strong evidence for a crucial role of the 24-hour rhythm of leptin sensitivity in the control of energy metabolism.
We furthermore demonstrated that mice with access to HFD exclusively during their leptin resistant phase (ZT21 – ZT3) displayed impairments in a variety of parameters that indicate metabolic health, such as compromised rhythms of locomotor activity, metabolic rate, and energy expenditure as well as increased circulating insulin levels. Restricting HFD exclusively to the leptin sensitive phase (ZT9 – ZT15), on the other hand, protected mice from the development of these severe metabolic impairments. To date it is still largely unknown whether HFD-induced development of metabolic diseases results from an increase in body fat content, diet composition or disrupted circadian rhythms. We observed these differences between TRF groups despite an identical reduction in body weight and plasma leptin levels in all TRF mice, suggesting that they are based on the time of food intake during the 24-hour rhythm of leptin sensitivity, but independent from factors such as body composition or HFD content. Nonetheless, all mice fed HFD displayed a reduction in the absolute values of average metabolic rate and energy expenditure relative to mice fed LFD, demonstrating that also the HFD itself affects energy metabolism. In conclusion, these results demonstrate that TRF is efficient in the reduction of body weight and the amelioration of metabolic health. However, our findings also highlight the importance of synchronising food intake with daily physiological rhythms to maintain metabolic health.
Taken together, this thesis identifies novel pathways that are involved in the neuroendocrine regulation of energy metabolism and provides new insights into the connection between physiological rhythms and the development of metabolic diseases
