Analysis of circadian pattern reveals tissue-specific alternative transcription in leptin signaling pathway

*Background*
It has been previously reported that most mammalian genes display a circadian oscillation in their baseline expression. Consequently, the phase and amplitude of each component of a signal transduction cascade has downstream consequences. 

*Results*
We report our analysis of alternative transcripts in the leptin signaling pathway which is responsible for the systemic regulation of macronutrient storage and energy balance. We focused on the circadian expression pattern of a critical component of the leptin signaling system, suppressor of cytokine signaling 3 (SOCS3). On an Affymetrix GeneChip 430A2 microarray, this gene is represented by three probe sets targeting different regions within the 3’ end of the last exon. We demonstrate that in murine brown adipose tissue two downstream 3’ probe sets experience circadian baseline oscillation in counter-phase to the upstream probe set. Such differences in expression patterns are a telltale sign of alternative splicing within the last exon of SOCS3. In contrast, all three probe sets oscillated in a common phase in murine liver and white adipose tissue. This suggests that the regulation of SOCS3 expression in brown fat is tissue specific. Another component of the signaling pathway, Janus kinase (JAK), is directly regulated by SOCS and has alternative transcript probe sets oscillating in counter-phase in a white adipose tissue specific manner.
 
*Conclusion*
We hypothesize that differential oscillation of alternative transcripts may provide a mechanism to maintain steady levels of expression in spite of circadian baseline variation

Circadian rhythms and hormonal homeostasis: Pathophysiological implications

Over recent years, a deeper comprehension of the molecular mechanisms that control biological clocks and circadian rhythms has been achieved. In fact, many studies have contributed to unravelling the importance of the molecular clock for the regulation of our physiology, including hormonal and metabolic homeostasis. Here we will review the structure, organisation and molecular machinery that make our circadian clock work, and its relevance for the proper functioning of physiological processes. We will also describe the interconnections between circadian rhythms and endocrine homeostasis, as well as the underlying consequences that circadian dysregulations might have in the development of several pathologic affections. Finally, we will discuss how a better knowledge of such relationships might prove helpful in designing new therapeutic approaches for endocrine and metabolic diseases

Impact of Sleep and Circadian Disruption on Energy Balance and Diabetes: A Summary of Workshop Discussions

A workshop was held at the National Institute for Diabetes and Digestive and Kidney Diseases with a focus on the impact of sleep and circadian disruption on energy balance and diabetes. The workshop identified a number of key principles for research in this area and a number of specific opportunities. Studies in this area would be facilitated by active collaboration between investigators in sleep/circadian research and investigators in metabolism/diabetes. There is a need to translate the elegant findings from basic research into improving the metabolic health of the American public. There is also a need for investigators studying the impact of sleep/circadian disruption in humans to move beyond measurements of insulin and glucose and conduct more in-depth phenotyping. There is also a need for the assessments of sleep and circadian rhythms as well as assessments for sleep-disordered breathing to be incorporated into all ongoing cohort studies related to diabetes risk. Studies in humans need to complement the elegant short-term laboratory-based human studies of simulated short sleep and shift work etc. with studies in subjects in the general population with these disorders. It is conceivable that chronic adaptations occur, and if so, the mechanisms by which they occur needs to be identified and understood. Particular areas of opportunity that are ready for translation are studies to address whether CPAP treatment of patients with pre-diabetes and obstructive sleep apnea (OSA) prevents or delays the onset of diabetes and whether temporal restricted feeding has the same impact on obesity rates in humans as it does in mice

Digital Signal Processing Reveals Circadian Baseline Oscillation in Majority of Mammalian Genes

In mammals, circadian periodicity has been described for gene expression in the hypothalamus and multiple peripheral tissues. It is accepted that 10%–15% of all genes oscillate in a daily rhythm, regulated by an intrinsic molecular clock. Statistical analyses of periodicity are limited by the small size of datasets and high levels of stochastic noise. Here, we propose a new approach applying digital signal processing algorithms separately to each group of genes oscillating in the same phase. Combined with the statistical tests for periodicity, this method identifies circadian baseline oscillation in almost 100% of all expressed genes. Consequently, circadian oscillation in gene expression should be evaluated in any study related to biological pathways. Changes in gene expression caused by mutations or regulation of environmental factors (such as photic stimuli or feeding) should be considered in the context of changes in the amplitude and phase of genetic oscillations

Impact of physiological rhythms on energy homeostasis in rodents

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

Cannabinoid CB1R Receptor Mediates Metabolic Syndrome in Models of Circadian and Glucocorticoid Dysregulation

The most recent projections in the growing obesity rates across the nation show an increase to 60% by the year 2030. These growing rates of obesity are paralleled by an increased rates of depressive conditions, anxiety, and sleep loss, often with environmental factors at the root of the cause. Stress and the stress response is a dynamic system that reflects one’s ability to cope with events, behaviorally or physiologically, as stressors occur over a lifetime. While many key players mediate the effects of stress exposure on disease outcomes including the sympathetic nervous system, parasympathetic, inflammatory cytokines and metabolic hormones, this dissertation focuses on glucocorticoids because of the extensive regulatory role they play in mounting the adaptative response to stress. Additionally, glucocorticoids act to regulate feeding and energy balance, and when not properly regulated, they can lead to increased weight gain, particularly the development of abdominal visceral fat. Independent from their role in the stress response, glucocorticoids are also secreted in a diurnal fashion as regulated by the master circadian (daily) clock in the suprachiasmatic nuclei ( SCN). Signaling in this regard is at least partially responsible for entraining circadian clocks outside of the SCN. Just as shifts in glucocorticoid exposure are associated with metabolic disturbances, disruption of circadian rhythms has also been linked to the development of obesity. However, it remains unclear how the two systems independently and/or collectively regulate energy balance. The first aim of this dissertation was to determine how disruptions in the environmental photoperiod impacts clock gene expression and if this environmental exposure affects circulating glucocorticoid levels. The second aim was to determine if increased exposure to a rhythmic corticosterone (CORT) disrupted circadian rhythms. Given that CORT imbalances result in circadian disruption and weight gain in both models, the third aim was to determine a mediator in the two systems that regulates CORT synthesis and affects metabolism. Recent studies have demonstrated that glucocorticoids possess the ability to increase the production and release of endocannabinoid molecules. Additionally, endocannabinoids are potent regulators of appetite, energy balance and metabolic processes through both central and peripheral regulation of feeding and metabolism, making the system an ideal candidate. Mice lacking the cannabinoid CB1 receptor were protected against all of these changes in metabolic function in both mouse models, indicating that endocannabinoid signaling is required for circadian disruption to promote obesity and metabolic syndrome through glucocorticoid regulation. These alterations are prevented by blocking the CB 1 receptor, not only globally, but also through targeted peripheral inhibition, suggesting that the endocannabinoid system mediates glucocorticoid induced metabolic syndrome through a predominantly peripheral mechanism. These data build upon previous findings that indicate the endocannabinoid system is required for diet-induced obesity. They further suggest that this system plays a much broader role in the regulation of metabolic processes, as well as acting as a mediator of changes in metabolic function in response to an array of stimuli, including environmental exposure, and not just diet composition. Research presented also aims to highlight the importance of studying different stress-like exposures in mouse models in order to fully characterize the human condition. Chronic environmental stressors, as in chronic disruption of the-light-dark-cycle, provide one such mean to study the impact of stress on a population. The environmental experience has been identified as a potential pathway linking neighborhood disadvantage and poor health, through the dysregulation of stress-related biological pathways

Comprehensive analysis of circadian periodic pattern in plant transcriptome

Abstract Background Circadian rhythm is a crucial factor in orchestration of plant physiology, keeping it in synchrony with the daylight cycle. Previous studies have reported that up to 16% of plant transcriptome are circadially expressed. Results Our studies of mammalian gene expression revealed circadian baseline oscillation in nearly 100% of genes. Here we present a comprehensive analysis of periodicity in two independent data sets. Application of the advanced algorithms and analytic approached already tested on animal data reveals oscillation in almost every gene of Arabidopsis thaliana. Conclusion This study indicates an even more pervasive role of oscillation in molecular physiology of plants than previously believed. Earlier studies have dramatically underestimated the prevalence of circadian oscillation in plant gene expression.</p

Regulation of Murine Physiology and Metabolism by the Adipocyte Circadian Clock through Sphingosine Kinase 1

The adipocyte, widely known for its large lipid droplets, is a crucial signaling cell at the crux of metabolism. Programs within the adipocyte itself may have massive impacts upon the body. For example, the internal cell-autonomous circadian clock within the adipocyte regulates such lipid droplet-associated processes as lipolysis (triacylglycerol breakdown) and lipogenesis (lipid uptake, triacylglycerol synthesis), which influence circulating lipids, thus impacting other organs. Circadian rhythms (molecular, metabolic, and behavioral patterning over a day) and lipid droplets are both highly evolutionarily conserved biological phenomena that also happen to be intertwined in the system about which we care today, the global human obesity pandemic. Excessive adiposity is a hallmark of obesity, and humans live in an extraordinarily artificially-lit world, which serves to disrupt circadian clocks, thereby disrupting metabolism. Characterized by their unique sphingosine backbone, sphingolipids, which may derive, in part, from the excess of adiposity and lipid free fatty acids, are well known to affect metabolic disease phenotypes. As the Cowart lab has shown in the past, and as shown in this work, sphingosine kinase 1 (SPHK1) and its enzymatic product sphingosine-1-phosphate (S1P) affect cellular signaling pathways that directly influence metabolism. SPHK1 is a lipid kinase which phosphorylates sphingosine (an N-acylated amino alcohol) to S1P, a bioactive autocrine and paracrine signaling lipid, to influence various processes including cellular survival, angiogenesis, endothelial permeability, immune cell trafficking, and oncogenesis. Since SPHK1 is highly involved in metabolism, such as glucose metabolism, and since metabolism is a circadian-governed process, we hypothesized that SPHK1 is involved in the regulation of the adipocyte circadian clock, which may impact circulating metabolites, such as glucose, fatty acids, and adipokines, thereby affecting weight gain and glucose tolerance. Using a wide variety of techniques and methodologies including RNA sequencing, mass spectrometry coupled with lipidomics and proteomics, chromatin immunoprecipitation (ChIP), protein co-immunoprecipitation, microscopy, gene and protein expression analysis, we conclude that novel properties of SPHK1 and S1P elicit changes in nuclear chromatin remodeling events, leading to circadian clock disruption and subsequent impairment of key metabolic physiological parameters in the mouse. To our knowledge, his is the first report of a lipid metabolite directly affecting the circadian clock