The adipose tissue, a major player in energy homeostasis in the body, has a complex mechanism of metabolism regulation, controlled both by internal rhythm and external stimuli, such as food intake. The effect of the circadian rhythm on the transcriptome of the adipose and liver has been described in animal models; however, studies examining diurnal or fasting effects or the effect of anti-obesity drugs on the transcriptome in the human adipose tissue are lacking.
The present study shows that the diurnal effect dominates the transcriptome of the human adipose tissue, with more than 25% of the transcribed genes being diurnally regulated. This finding is consistent with observed circadian regulation in adipose in animal models, in which up to 50% of the genes are under circadian control [5, 7, 11]. Moreover, the results demonstrated robust regulation of the core clock gene PER1 and of genes encoding for ribosome processing and biogenesis and inflammatory processes. Ribosome biogenesis genes were on the diurnal incline, with levels rising by afternoon and remaining constant until evening. Ribosome biogenesis is an indicator of cellular activity and, in this case, most likely driven by the AKT/PI3K/mTOR pathway . A number of genes encode enzymes in glucose, mannose and fructose metabolism (PFKFB3, FUK, MPI, PFKM), with high expression levels in the morning and a decline in the afternoon through the evening, following the trend of PER1. Conversely, "fuel accumulation" genes, such as those involved in cholesterol biosynthesis (HMGCR, HMGSC1), LDL receptor (LDLR), and glucose transport (GLUTS 1, 3, 5 and 14) have low levels in the morning and rise in the afternoon. Interestingly, there was no observed correlation between the more typical lipogenesis or transporter genes, such as fatty acid synthase (FASN) or GLUTs 2 and 4, and PER1 expression. These genes may be regulated in a more acute fashion by external stimulation, such as insulin or cholesterol, or may not be sensitive to diurnal regulation in the adipose tissue of mildly obese subjects.
Many genes encoding for cytokines and other inflammation-related proteins were also diurnally regulated. The mRNA levels of this set of genes were inversely correlated with PER1 expression, with expression levels increasing dramatically from the morning through the afternoon and being highest in the evening. These genes had among the highest amplitudes of change, from 2-fold (IL-10) to 20-fold (IL-6). Whereas studies have previously shown that inflammation-related proteins, such as PAI-1, IL-6 and TNFα, were diurnally regulated [12, 28], the present result adds several new cytokines, including PTX3, IL1, IL10, GRO1, GRO2, CCL6, TGFA and CCL7 to the set of known diurnally regulated genes. Many cytokines, such as IL-6 and IL-8 and MCP-1, have been implicated in cardiovascular risk; the present study demonstrated that both IL-6 and IL-8 were significantly, but inversely, correlated with PER1. The observed associations between these pro-inflammatory genes with the diurnal rhythm warrant further investigation.
To further characterize the physiology of the diurnal change in the human adipose, an unbiased in silico search for compound signatures common with the diurnally regulated genes in our study was performed using the publicly available Connectivity Map database. Significant overlap was observed with the AKT/PI3K/mTOR pathway inhibitors, leading to the hypothesis that a signature elicited by insulin or other growth factors would also overlap with the diurnal signature. To test this hypothesis, we used a set of genes that were regulated by treatment of growth factors such as EGF, b-FGF, IGF1, Insulin or Heregulin in MCF7 and HT29 cell lines (Additional file 9; manuscript submitted). As expected, the growth factor pathway genes were correlated with PER1 and the correlations were in the same direction as that of the diurnal set. Moreover, the growth factor gene set linked to the same growth inhibitors from the Connectivity Map query.
The connection between the AKT/PI3K/mTOR pathway and the diurnally regulated adipose tissue is intriguing. Several studies have linked the AKT/PI3K/mTOR pathway to obesity and, independently, the circadian rhythm to metabolic syndrome [16, 30, 31]. A key kinase in the mTOR pathway is S6K. The S6K -/- mouse is resistant to diet-induced obesity, having adipocytes that do not accumulate lipids . The mTOR pathway is strongly upregulated during adipogenesis [31, 32]. The CLOCK mutant mouse has metabolic syndrome . Regulated by AKT and a key player in the AKT/PI3K/mTOR pathway, glycogen synthase kinase 3 beta (GSK-3), the critical checkpoint for glycogen synthesis, is linked to the circadian rhythm . Modulation of GSK-3, also known as shaggy, alters circadian rhythms in Drosophila and affects clock genes in mammalian cells [34, 35]. The findings of the present study are consistent with the connection between the mTOR pathway and the link between circadian rhythm and glucose metabolism. Several cancer drugs that target growth factor pathways might "reverse" the circadian pattern, thus preventing adipose from going into lipid accumulating/anabolic state in the evening. This hypothesis is consistent with the reported side effects of sirolimus, a drug with a significant negative association with circadian rhythm and that leads to hyperlipidemia and accumulation of fatty acids in circulation [36, 37], possibly owing to the very high doses necessary, which may prevent the anabolic state of the adipose. Data from various F2 mouse crosses also show that mTOR is causal for obesity traits . Taken together, these observations suggest that anti-cancer drugs, in appropriate doses, may be useful anti-obesity compounds.
Consistent with the observations on the tissue level, the addition of glucose to a rodent cell line led to the down regulation of PER1 and induction of circadian oscillations . In the same model system, oscillations have been induced by the addition of growth factors or prolonged activation of MAPK pathway, and stalled by MEK inhibitors . In addition, BMAL and CLOCK have involvement in glucose homeostasis . These results, together with the findings from the present study, provide support for an association of circadian rhythm with growth factor signaling and metabolic effects.
Another interesting compound uncovered by the in silico query of the Connectivity Map was resveratrol, a natural activator of SIRT1 – a circadian deacetylase for core clock components, countering the effect of CLOCK, shown to have histone acetylation activity [42, 43]. SIRT1 is also linked to metabolic disease . This result provides additional support for the link between diurnal rhythm and metabolic output. In addition, the diurnal signature clearly overlapped (p = 1.16 × 10-13) with a large set of key genes that form an adipose module in a gene-gene correlation network that tested causal for various metabolic endpoints, such as obesity, diabetes and cardiovascular disease . This underscores that genes in the adipose diurnal signature can be mined for drug targets against obesity and other metabolic phenotypes. One of the most correlated PER1 genes in the adipose was ZNF145 (also known as PLZF), which drives metabolic syndrome in rats and affects the transcription of the prorenin/renin receptor [24, 46]. A recently implicated gene in humans for obesity, FTO, is also part of the PER1 signature .
The physiological changes associated with the diurnal variation of the human adipose transcriptome are important to understand. It is reasonable that humans, like other organisms that live according to the light-dark cycle imposed by earth's rotation, would evolve to compartmentalize energy metabolism in synchrony with diurnal rhythm. We hypothesize that diurnal rhythm in human adipose underlies the transition from a catabolic, energy-releasing state in the morning to an anabolic, energy-storing state in the evening.
A phase shift in circadian rhythm induced by restricted feeding has been reported in animal studies . Similarly, the present study showed that both the fasting state and sibutramine were part of the diurnal signature, indicating that these two perturbations had an effect on the metabolism of the adipose tissue. Both interventions induced a temporal delay in the diurnal rhythm, thereby extending the catabolic state of the adipose (into the afternoon with fasting and into the evening with sibutramine). The delay observed in the fasting arm ended by the evening, likely owing to the fact that subjects were fed in the afternoon. However, the delay caused by sibutramine was evident at the last time point, even after feeding, indicating that sibutramine was still actively affecting the diurnal genes. One of the most differentially down regulated genes in the sibutramine versus the fasted arm at the 10 hour time point was chemokine (C-X-C motif) ligand 1 (CXCL1), a secreted cytokine involved in numerous inflammatory pathways. CXCL1 is also known as growth-regulated oncogene-alpha (GRO-alpha) and is involved in many tumor types as an oncogene [48, 49].
Differences between the regulation of clock genes in humans versus rodents have been observed. Contrary to the results in human adipose in this study, expression of PER1 mRNA in rodents increased from the morning through the evening . In addition, the effect of fasting and sibutramine in human adipose tissue was subtle; again, quite different than in rodents, for which restricted feeding produces profound effects on the peripheral clock . However, compared with the present study conducted in overweight to mildly obese humans in the course of one day, many of the rodent studies were conducted in lean mice and the restricted feeding regimen was conducted over many days, potentially confounding the comparison between rodents and humans. Rhythmic expression of clock genes is attenuated in the perigonadal adipose tissues of obese KK mice and obese, diabetic KK-Ay mice, indicating that obesity and disease state are intricately linked to the circadian rhythm . To further investigate the differences between rodent models and humans and the association between obesity and circadian rhythm in humans, a similar study in lean and morbidly obese individuals could be conducted. Nevertheless, despite the limitations noted, the major finding is that many genes in the peripheral tissues, such as the adipose in both rodents and humans, exhibit rhythmic expression. The circadian output genes are also linked to metabolism in both species and are affected by such stimuli as restricted feeding. Rodent studies, examining white and brown adipose tissue, liver and skeletal muscle, also showed the number of genes under circadian regulation ranging from 3% to 26%, suggesting that a large proportion of the transcriptome is under circadian control [5, 10]. The estimation from the current study is closer to the upper bound of what has been observed in rodents. This could simply reflect the differences in the experimental designs and statistical power to detect changes. On the other hand, it could be attributed to the dominant role of the adipose in driving peripheral clocks in overweight individuals. Clocks in peripheral tissues can be entrained by feeding . One can speculate that feeding patterns in humans may play a substantial role in the synchronization of SCN-controlled and food entrainable oscillations. This synchronization may lead to more efficient energy utilization by adipose and, in turn, may explain the effect of clock-related genes, such as Nocturnin, on resistance to diet-induced obesity . Understanding cross-species similarities and differences is necessary for a deeper understanding of how circadian rhythm affects physiology on the whole.