Molecular clock assessment in different human cell types

Exploring molecular clock in human peripheral organs represents a very non-trivial technical challenge due to the obvious reasons [126].

The use of blood cells could assess the human physiological clock. Determining human internal body time could provide breakthroughs in the field of chronopharmacology, since it’s crucial to administrate a medication at a proper time to enhance its efficiency. Minami et al have developed a new method to evaluate internal body time using different mass-spectrometry: gas chromatography mass spectrometry, liquid chromatography mass spectrometry and capillary electrophoresis mass spectrometry in mice. By combining these three methods, they were able to establish a molecular timetable based on blood metabolome analysis [127]. The same group applied this method to human blood sample analyses to estimate the internal body time and the delay caused by enforced sleep-wake cycle of each individual. The oscillating metabolites were identified [128]. Human transcriptome has been performed under forced-desynchrony protocol by Archer et al. They found that central clock remained rhythmic while blood peripheral transcriptome became arrhythmic [129].

In 1998, Balsalobre et al established that cultured rat fibroblasts possessed an internal clock that could be assessed in cells following in vitro synchronization by high concentration of horse serum [130]. E. Nagoshi et al demonstrated that NIH3T3 fibroblasts had a self-sustained and cell-autonomous circadian rhythm and could be synchronized by adding serum [131].

The use of fibroblasts from humans then appeared as an easy model to assess peripheral circadian rhythm.

43 The period length could be measured by bioluminescence of reporter gene luciferase driven by Bmal1 promoter in the human skin fibroblasts, blood cells and keratinocytes (Figure 7).

Using this construct, circadian rhythm was recorded from the fibroblasts of 19 volunteers by Steven Brown and colleagues, and they found that period length was specific to each individual with a standard deviation of 48 minutes among the subjects. The wheel-running period reported from wild-type and mutant mice for circadian genes corresponded to the period length assessed in tail fibroblasts [132].

Furthermore, the circadian rhythm was assessed in 28 subjects with extreme chronotypes (early, late) in human skin fibroblasts by the same approach. The period length was different between the two groups, suggesting that the oscillation pattern measured in the skin fibroblasts synchronised in vitro reflects the in vivo rhythm. [133]. Additional studies by the same group supported the concept that period length assessed in peripheral clocks corresponds to the physiological period length. Pagani and Brown validated that the period length assessed in human skin fibroblasts using Bmal1-luciferase reporter gene was directly proportional to the period length measured in vivo using saliva melatonin [134]. Moreover, it was demonstrated that in a population of 35 patients with PER3 VNTR polymorphism, in vivo period length assessed by plasma melatonin was shorter than in vitro periods obtained from skin fibroblasts and measured by bioluminescence of Bmal1-luciferase construct [135].

In addition, the effect of serum factors on the modulation of the circadian system has been tested in humans. The skin fibroblasts from young and older subjects were collected and circadian rhythm was assessed using Bmal1-luciferase construct. No significant difference of the circadian parameters was found between these two groups of subjects, but the period length was shortened and phase shifted to earlier when serum from older individuals was applied on cells from young subjects [136].

44 Figure 7. Thyrocytes, skeletal muscle myotubes, islets and primary fibroblasts synchronized in vitro displayed circadian oscillations assessed by bioluminescence [126].

The existence of a functional molecular clock has also been demonstrated in different human cells like in adipocytes [137], blood mononuclear cells [138], keratinocytes [139], pancreatic islets [111] and human primary skeletal myotubes [140] (Figure 7, [126]). Interestingly, our group has shown that human thyrocytes exhibited circadian oscillations, which were disrupted in thyroid malignancies and could thus be used as potential diagnostic biomarkers for thyroid malignancies [141,142].

In rodents, the peripheral clocks have been found in almost all organs. Associations between altered molecular clock and pathologies are constantly explored. In humans, it is more difficult to look at a direct link between an altered expression of clock genes and the occurrence of the pathology. That is why genetic analyses using SNP and studies of –omics in

45 populations with different characteristics and diseases are evolving [143]. Further investigations are needed to explore the molecular mechanisms implying the circadian clock.

For this project, healthy volunteers, T2D or obese patients have been recruited. Human fibroblasts derived from skin have been cultured. The cells were transduced with lentivectors containing luciferase gene driven by circadian promoter Bmal1 or Per2. The oscillations profiles were obtained by bioluminescence and the circadian parameters were compared between the groups. Blood collection was performed on these subjects to allow correlation analyses with the period length. The following chapters present the precise methodology and the results of this project.

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47 4. HYPOTHESIS

Studies in rodent models suggest that on one hand, the genetic models of core-clock gene knockouts are prone to obesity and T2D. On the other hand, high fat diet induced obesity impacts on the central and peripheral clock. Based on the accumulating evidence on the interconnection between the circadian clock, glucose and lipid metabolism, and metabolic diseases in rodents, and on epidemiological studies in humans, we hypothesize that the circadian oscillator might be altered in human subjects upon metabolic diseases.

Based on the studies in rodents suggesting that high fat diet induced obesity alters molecular properties of the peripheral oscillators, we asked whether in humans the molecular clock might bear distinct circadian characteristics in cells derived from T2D and obese patients, if compared to healthy counterparts.

Furthermore, in view of the likely possibility of deleterious effects of high amount of glucose and fatty acid on the circadian oscillations, we queried whether the application of these components in vitro on human primary cells may affect cellular oscillation profiles.

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49 5. OBJECTIVE

The overall objective of this study is to characterize the molecular properties of the circadian oscillators operative in human primary skin fibroblasts established from healthy volunteers, in comparison to those derived from obese, and T2D (obese and non-obese) patients. We set as a goal providing a comprehensive picture of the cellular mechanisms connecting circadian clock with the metabolic disease in humans.

Specifically, we aimed at:

1. Getting the new insights into peripheral clock function upon obesity and T2D in human subjects. To this end we aimed at obtaining and analyzing the circadian profiles of primary skin fibroblasts established from healthy, obese, T2D non-obese, and T2D obese individuals.

Potential association of oscillation profiles of these cellular clocks with the metabolic diseases (obesity and T2D) are evaluated.

2. Assessing the impact of serum factors on the circadian rhythm in human primary fibroblasts. To this end, the sera from healthy subjects will be applied to the T2D patient skin fibroblasts and vice versa.

3. Determining the impact of glucotoxicity and lipotoxicity on the circadian profiles in these groups of patients based on our in vitro cellular system.

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51 6. MATERIALS AND METHODS