BackgroundDiurnal behavior in humans is governed by the period length of a circadian clock in the suprachiasmatic nuclei of the brain hypothalamus. Nevertheless, the cell-intrinsic mechanism of this clock is present in most cells of the body. We have shown previously that for individuals of extreme chronotype (“larks” and “owls”), clock properties measured in human fibroblasts correlated with extreme diurnal behavior.Methodology/Principal FindingsIn this study, we have measured circadian period in human primary fibroblasts taken from normal individuals and, for the first time, compared it directly with physiological period measured in vivo in the same subjects. Human physiological period length was estimated via the secretion pattern of the hormone melatonin in two different groups of sighted subjects and one group of totally blind subjects, each using different methods. Fibroblast period length was measured via cyclical expression of a lentivirally delivered circadian reporter. Within each group, a positive linear correlation was observed between circadian period length in physiology and in fibroblast gene expression. Interestingly, although blind individuals showed on average the same fibroblast clock properties as sighted ones, their physiological periods were significantly longer.Conclusions/SignificanceWe conclude that the period of human circadian behaviour is mostly driven by cellular clock properties in normal individuals and can be approximated by measurement in peripheral cells such as fibroblasts. Based upon differences among sighted and blind subjects, we also speculate that period can be modified by prolonged unusual conditions such as the total light deprivation of blindness.

Although overt diurnal rhythms of behavior do not begin until well after birth, molecular studies suggest that the circadian clock may begin much earlier at a cellular level: mouse embryonic fibroblasts, for example, already possess robust clocks. By multiple criteria, we found no circadian clock present in mouse embryonic stem cells. Nevertheless, upon their differentiation into neurons, circadian gene expression was observed. In the first steps along the pathway from ES cells to neurons, a neural precursor cell (NPC) line already showed robust circadian oscillations. Therefore, at a cellular level, the circadian clock likely begins at the very earliest stages of mammalian development.

The mechanisms by which feeding and fasting drive rhythmic gene expression for physiological adaptation to daily rhythm in nutrient availability are not well understood. Here we show that, upon feeding, the RNA-binding protein NONO accumulates within speckle-like structures in liver cell nuclei. Combining RNA-immunoprecipitation and sequencing (RIP-seq), we find that an increased number of RNAs are bound by NONO after feeding. We further show that NONO binds and regulates the rhythmicity of genes involved in nutrient metabolism post-transcriptionally. Finally, we show that disrupted rhythmicity of NONO target genes has profound metabolic impact. Indeed, NONO-deficient mice exhibit impaired glucose tolerance and lower hepatic glycogen and lipids. Accordingly, these mice shift from glucose storage to fat oxidation, and therefore remain lean throughout adulthood. In conclusion, our study demonstrates that NONO post-transcriptionally coordinates circadian mRNA expression of metabolic genes with the feeding/fasting cycle, thereby playing a critical role in energy homeostasis.

Various lines of evidence suggest a mechanistic role for altered cAMP-CREB (cAMP response element - binding protein) signaling in depressive and affective disorders. However, the establishment and validation of human inter-individual differences in this and other major signaling pathways has proven difficult. Here, we describe a novel lentiviral methodology to investigate signaling variation over long periods of time directly in human primary fibroblasts. On a cellular level, this method showed surprisingly large inter-individual differences in three major signaling pathways in human subjects that nevertheless correlated with cellular measures of genome-wide transcription and drug toxicity. We next validated this method by establishing a likely role for cAMP-mediated signaling in a human neuroendocrine response to light - the light-dependent suppression of the circadian hormone melatonin - that shows wide inter-individual differences of unknown origin in vivo. Finally, we show an overall greater magnitude of cellular CREB signaling in individuals with bipolar disorder, suggesting a possible role for this signaling pathway in susceptibility to mental disease. Overall, our results suggest that genetic differences in major signaling pathways can be reliably detected with sensitive viral-based reporter profiling, and that these differences can be conserved across tissues and be predictive of physiology and disease susceptibility.

1 This study aimed to evaluate the effects of phosphodiesterase (PDE) inhibitors and currently prescribed anti-asthma drugs for their ability to inhibit inflammatory cell activation in vitro. 2 Alveolar macrophages and eosinophils were isolated from the bronchoalveolar lavage (BAL) fluid of ovalbumin (Ovalb)-sensitized guinea-pigs. Opsonized zymosan (OZ) and PAF stimulated leukotriene B4 (LTB4) release from eosinophils was measured by radioimmunoassay. Ovalb-induced superoxide generation was measured by reduction of cytochrome C. 3 Monocytes were separated from human peripheral venous blood and mast cells were dispersed from human lung fragments. Lipopolysaccharide (LPS)-induced tumour necrosis factor-a (TNF-a) release from monocytes was measured by ELISA and anti-IgE stimulated histamine release from mast cells was measured by a radioenzymatic method. 4 The fl2 agonist, salbutamol inhibited TNF-a release from monocytes and histamine release from mast cells whilst having no effect on eosinophil-derived LTB4 release or macrophage superoxide generation. 5 The PDE 3 inhibitor, milrinone produced a concentration-related inhibition of TNF-a release from monocytes which achieved statistical significance at 10-5 M but inhibited LTB4 release from eosinophils and superoxide generation from macrophages only at the highest concentration (10-3 M) examined. Milrinone had no effect on histamine release from mast cells. 6 The selective PDE 4 inhibitors, denbufylline and rolipram and the corticosteroid, beclomethasone produced a concentration-related inhibition of LTB4 release from eosinophils, TNF-ax release from monocytes and superoxide generation from alveolar macrophages whilst having no effect on histamine release from mast cells. 7 The mixed PDE 3/4 inhibitor, benzafentrine produced a concentration-related inhibition of LTB4 release from eosinophils, TNF-a release from monocytes, superoxide generation from alveolar macrophages and histamine release from mast cells. 8 In conclusion these data clearly show that both established anti-asthma medication as well as PDE inhibitors have the potential to inhibit inflammatory cell activation in vitro but that the anti-secretory actions of fl2 agonists, corticosteroids and PDE inhibitors are distinct.

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