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

The clock genes Period 2 and Cryptochrome 2 differentially balance bone formation

Background: Clock genes and their protein products regulate circadian rhythms in mammals but have also been implicated in various physiological processes, including bone formation. Osteoblasts build new mineralized bone whereas osteoclasts degrade it thereby balancing bone formation. To evaluate the contribution of clock components in this process, we investigated mice mutant in clock genes for a bone volume phenotype. Methodology/Principal Findings: We found that Per2Brdm1 mutant mice as well as mice lacking Cry2-/- displayed significantly increased bone volume at 12 weeks of age, when bone turnover is high. Per2Brdm1 mutant mice showed alterations in parameters specific for osteoblasts whereas mice lacking Cry2-/- displayed changes in osteoclast specific parameters. Interestingly, inactivation of both Per2 and Cry2 genes leads to normal bone volume as observed in wild type animals. Importantly, osteoclast parameters affected due to the lack of Cry2, remained at the level seen in the Cry2-/- mutants despite the simultaneous inactivation of Per2. Conclusions/Significance: This indicates that Cry2 and Per2 affect distinct pathways in the regulation of bone volume with Cry2 influencing mostly the osteoclastic cellular component of bone and Per2 acting on osteoblast parameters

Rhythmic Diurnal Gene Expression in Human Adipose Tissue From Individuals Who Are Lean, Overweight, and Type 2 Diabetic

OBJECTIVE Previous animal studies suggest a functional relationship between metabolism, type 2 diabetes, and the amplitude of daily rhythms in white adipose tissue (WAT). However, data interpretation is confounded by differences in genetic background and diet or limited sampling points. We have taken the novel approach of analyzing serial human WAT biopsies across a 24-h cycle in controlled laboratory conditions.RESEARCH DESIGN AND METHODS Lean (n = 8), overweight/obese (n = 11), or overweight/obese type 2 diabetic (n = 8) volunteers followed a strict sleep-wake and dietary regimen for 1 week prior to the laboratory study. They were then maintained in controlled light-dark conditions in a semirecumbent posture and fed hourly during wake periods. Subcutaneous WAT biopsies were collected every 6 h over 24 h, and gene expression was measured by quantitative PCR.RESULTS Lean individuals exhibited significant (P 0.05) of increased body weight or type 2 diabetes on rhythmic gene expression.CONCLUSIONS The robust nature of these rhythms and their relative phasing indicate that WAT now can be considered as a peripheral tissue suitable for the study of in vivo human rhythms. Comparison of data between subject groups clearly indicates that obesity and type 2 diabetes are not related to the amplitude of rhythmic WAT gene expression in humans maintained under controlled conditions. Diabetes 60:1577-1581, 201

Nocturnin Expression Is Induced by Fasting in the White Adipose Tissue of Restricted Fed Mice

The relationship between circadian clocks and metabolism is intimate and complex and a number of recent studies have begun to reveal previously unknown effects of food and its temporal availability on the clock and the rhythmic transcriptome of peripheral tissues. Nocturnin, a circadian deadenylase, is expressed rhythmically in a wide variety of tissues, but we report here that Nocturnin expression is arrhythmic in epididymal white adipose tissue (eWAT) of mice housed in 12∶12 LD with ad libitum access to food. However, Nocturnin expression becomes rhythmic in eWAT of mice placed on restricted feeding. We show here that Nocturnin's rhythmic expression pattern is not dependent upon feeding, nor is it acutely induced by feeding in the liver or eWAT of ad libitum fed mice. However, Nocturnin is acutely induced by the absence of the expected meal in eWAT of restricted fed mice. A rise in cAMP levels also induces Nocturnin expression, suggesting that Nocturnin's induction in eWAT by fasting is likely mediated through the same pathways that activate lipolysis. Therefore, this suggests that Nocturnin plays a role in linking nutrient sensing by the circadian clock to lipid mobilization in the adipocytes

Daily Rhythms of Plasma Melatonin, but Not Plasma Leptin or Leptin mRNA, Vary between Lean, Obese and Type 2 Diabetic Men

Melatonin and leptin exhibit daily rhythms that may contribute towards changes in metabolic physiology. It remains unclear, however, whether this rhythmicity is altered in obesity or type 2 diabetes (T2DM). We tested the hypothesis that 24-hour profiles of melatonin, leptin and leptin mRNA are altered by metabolic status in laboratory conditions. Men between 45–65 years old were recruited into lean, obese-non-diabetic or obese-T2DM groups. Volunteers followed strict sleep-wake and dietary regimes for 1 week before the laboratory study. They were then maintained in controlled light-dark conditions, semi-recumbent posture and fed hourly iso-energetic drinks during wake periods. Hourly blood samples were collected for hormone analysis. Subcutaneous adipose biopsies were collected 6-hourly for gene expression analysis. Although there was no effect of subject group on the timing of dim light melatonin onset (DLMO), nocturnal plasma melatonin concentration was significantly higher in obese-non-diabetic subjects compared to weight-matched T2DM subjects (p<0.01) and lean controls (p<0.05). Two T2DM subjects failed to produce any detectable melatonin, although did exhibit plasma cortisol rhythms comparable to others in the group. Consistent with the literature, there was a significant (p<0.001) effect of subject group on absolute plasma leptin concentration and, when expressed relative to an individual’s 24-hour mean, plasma leptin showed significant (p<0.001) diurnal variation. However, there was no difference in amplitude or timing of leptin rhythms between experimental groups. There was also no significant effect of time on leptin mRNA expression. Despite an overall effect (p<0.05) of experimental group, post-hoc analysis revealed no significant pair-wise effects of group on leptin mRNA expression. Altered plasma melatonin rhythms in weight-matched T2DM and non-diabetic individuals supports a possible role of melatonin in T2DM aetiology. However, neither obesity nor T2DM changed 24-hour rhythms of plasma leptin relative to cycle mean, or expression of subcutaneous adipose leptin gene expression, compared with lean subjects

Characterizing Dynamic Changes in the Human Blood Transcriptional Network

Gene expression data generated systematically in a given system over multiple time points provides a source of perturbation that can be leveraged to infer causal relationships among genes explaining network changes. Previously, we showed that food intake has a large impact on blood gene expression patterns and that these responses, either in terms of gene expression level or gene-gene connectivity, are strongly associated with metabolic diseases. In this study, we explored which genes drive the changes of gene expression patterns in response to time and food intake. We applied the Granger causality test and the dynamic Bayesian network to gene expression data generated from blood samples collected at multiple time points during the course of a day. The simulation result shows that combining many short time series together is as powerful to infer Granger causality as using a single long time series. Using the Granger causality test, we identified genes that were supported as the most likely causal candidates for the coordinated temporal changes in the network. These results show that PER1 is a key regulator of the blood transcriptional network, in which multiple biological processes are under circadian rhythm regulation. The fasted and fed dynamic Bayesian networks showed that over 72% of dynamic connections are self links. Finally, we show that different processes such as inflammation and lipid metabolism, which are disconnected in the static network, become dynamically linked in response to food intake, which would suggest that increasing nutritional load leads to coordinate regulation of these biological processes. In conclusion, our results suggest that food intake has a profound impact on the dynamic co-regulation of multiple biological processes, such as metabolism, immune response, apoptosis and circadian rhythm. The results could have broader implications for the design of studies of disease association and drug response in clinical trials

Adipocyte extracellular matrix composition, dynamics and role in obesity

The central role of the adipose tissue in lipid metabolism places specific demands on the cell structure of adipocytes. The protein composition and dynamics of the extracellular matrix (ECM) is of crucial importance for the functioning of those cells. Adipogenesis is a bi-phasic process in which the ECM develops from a fibrillar to a laminar structure as cells move from the commitment phase to the growth phase characterized by storage of vast amounts of triglycerides. Mature adipocytes appear to spend a lot of energy on the maintenance of the ECM. ECM remodeling is mediated by a balanced complement of constructive and destructive enzymes together with their enhancers and inhibitors. ECM remodeling is an energy costing process regulated by insulin, by the energy metabolism, and by mechanical forces. In the obese, overgrowth of adipocytes may lead to instability of the ECM, possibly mediated by hypoxia