Impact of FFA on the circadian oscillations of human fibroblasts

Type 2 diabetic group

7.9.1. Impact of FFA on the circadian oscillations of human fibroblasts

To explore the effects of free fatty acids on the circadian rhythm, we tested the most common saturated fatty acid palmitate (PA), oleic acid (OA) a monounsaturated long chain fatty acid, and linoleic acid (LA), a Ω6 polyunsaturated fatty acid. The oscillation profiles of human fibroblasts were analysed when the cells were incubated with FFAs in the recording medium during 6 days.

Compared to untreated cells, the presence of a fatty acid seems to lower the oscillation amplitude, (Figure 29) although in the absence of normalization for the cell number this result requires further validation. The period lengths of the cells incubated with isolated fatty acids were not different as compared to the control condition (control period length mean ± SD:

25.9 ± 1.8, PA: 24.6 ± 1.33, p=0.310; OA: 24.1 ± 1.42, p=0.151; LA: 24.9 ± 0.51, p=0.222).

Figure 29. Incubation of human fibroblasts with fatty acids. Palmitic acid (PA), oleic acid (OA) and linoleic acid (LA) at a final concentration of 100 μM were added in the recording medium after dexamethasone synchronization. Cells from healthy subject (H9) were transduced with Bmal1-luciferase lentivector. 15% of FCS was added in the white medium for the control condition. Each condition was performed in duplicate dishes.

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117 The effects of palmitate conjugated with BSA and of oleate on the cellular rhythms were assessed in different conditions of incubation with FFAs, in order to mimic the effect of chronic lipotoxicity.

We found that the period length was longer than the control condition when the cells were incubated with palmitate in the recording medium (p=0.002). Compared to the untreated cells, fibroblasts incubated with BSA had a lower period length (p=0.026) and showed a phase delay of an average of 3 hours 9 minutes. This phase delay was attenuated when the cells were incubated with palmitate in the recording medium (1 hour and 42 minutes) or in the preincubation medium (51 minutes). (Figure 30). Although the palmitate altered the oscillation period of the skin fibroblasts in comparison to the control condition, given the opposite effect of BSA on the period length it is difficult to draw the solid conclusions from these experiments.

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118 B.

Figure 30. Oscillation profiles and period lengths of cells incubated with oleate or palmitate. A.

Human fibroblasts derived from two healthy volunteers (H4, H6) and transduced with Bmal1-luc lentivectors were incubated for 48 hours with 200uM of palmitate, oleate, or BSA, used as the control condition for palmitate, since palmitate is conjugated with BSA as following:

BSA -/-: Control condition, the standard culture medium containing BSA was added 48 hours before the synchronization with dexamethasone, and replaced with the white recording medium containing BSA and no fatty acids during the recording period.

BSA palmitate -/+: The standard culture medium containing BSA was added 48 hours prior to the synchronization, and replaced with the recording medium containing palmitate conjugated with BSA.

Palmitate BSA +/-: The standard culture medium containing Palmitate was added 48 hours prior to the synchronization, and replaced with the recording medium containing BSA.

Palmitate +/+: The standard culture medium containing Palmitate was added 48 hours prior to the synchronization, and replaced with the recording medium containing palmitate conjugated with BSA.

Medium -/-: Control condition, the standard culture medium was added 48 hours before the synchronization, and replaced again with the standard recording medium.

Medium oleate -/+: The standard culture medium was added 48 hours before the synchronization, and replaced with the recording medium containing oleate.

Oleate medium +/-: The standard culture medium containing oleate was added in the medium 48 hours prior to the synchronization, and replaced with standard recording medium.

Oleate +/+: The standard culture medium containing oleate was added in the medium 48 hours prior to the synchronization, and replaced with recording medium containing oleate.

B. Period length measured for each condition. Data are represented in mean ± SEM.

119 7.9.2. Lipid mixtures added in the recording medium do not affect the circadian

oscillations

To investigate the potential of lipid mixture to modulate the circadian rhythm in human fibroblasts, cells from healthy volunteers were incubated for 5 days in the recording medium containing emulsions of Ω3 and Ω6 fatty acids. We tested the effects of two concentrations (10 and 50 mg/l) of Omegaven® and Lipoven® that are mixtures of Ω3 (EPA and DHA) and Ω6 (soya oil) respectively. No differences of period length were found neither for cells incubated with Omegaven® nor with Lipoven® (Figure 31).

A. B.

Figure 31. Incubation of human fibroblasts with Omegaven® or Lipoven®. A. Omegaven® or Lipoven® at a final concentration of 10 or 50 mg/l was added to the recording medium, following dexamethasone synchronization. Cells from healthy subject (H4) were transduced with Bmal1-luciferase lentivector. 15% of FCS was added to the white medium for the control condition. Each condition was assessed in duplicate dishes. B. Circadian period length for each condition calculated based on Bmal1-luciferase bioluminescence profiles. Data are presented in mean ± SD.

120 7.9.3. Effect of gluco-lipotoxicity on circadian oscillations of human skin

fibroblasts

The high concentration of glucose concentration or the addition of FFAs or lipid mixture to the cell culture medium alone did not affect significantly the oscillation profiles of human fibroblasts. We thus tested whether a combination of both FFAs and glucose could affect the circadian oscillations. Cells established from three healthy volunteers were incubated in the presence of three different concentrations of glucose in combination with oleate, palmitate, BSA or standard medium. The addition of oleate did not affect the period length in the presence of different concentrations of glucose in the medium (Figure 32B). Cells incubated with palmitate in medium containing 1 g/l or 2 g/l of glucose tended to a longer period length compared to BSA (p=0.05) (Figure 32B). However, the addition of palmitate affected the viability of the cells in the presence of three different concentrations of glucose but it was not statistically different compared to BSA (Figure 32C-D).

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Palmitate 1g/l Palmitate 2g/l Palmitate 4.5g/l

121 B.

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Mortality rates (%)

122 Figure 32. Incubation of human fibroblasts with combined glucose and FFA at different concentrations.

Human fibroblasts derived from three healthy volunteers (H5, H6, H9) and transduced with Bmal1-luc lentivectors were incubated with palmitate 500 μM, oleate 160 μM, or BSA, used as the control condition for palmitate. The compounds were incubated in medium with 1 g/l, 2 g/l or 4.5 g/l glucose 48 hours prior to the synchronization and in the recording medium. A. Oscillation profiles of cells subjected to different glucose concentration and fatty acids. B. Period length measured for each condition. Data are represented in mean ± SEM. C. Mortality rates assessed by trypan blue five days after synchronization. D. Pictures of human fibroblasts affected by palmitate. Upper left panel: not treated cells in standard medium (1 g/l glucose) before synchronization; upper right: not treated cells in standard medium (1 g/l glucose) five days after synchronization; lower left: cells before palmitate incubation in standard medium (1 g/l glucose); lower right: cells after palmitate incubation in standard medium (1 g/l glucose) five days after synchronization.

123 8. DISCUSSION AND CONCLUSION

Our work aimed at studying molecular properties of the circadian clock in human subjects upon metabolic diseases such as obesity and T2D. Our experimental approach is based on assessment of the molecular clocks in primary human skin fibroblasts derived from the skin biopsies and synchronized in vitro. This approach proved highly instrumental for studying human peripheral clocks in physiology and different pathologies [115,141,162]. While no significant differences in the oscillator makeup were detected between the groups of healthy, obese and T2D subjects, a strong inverse correlation between the levels of glycated hemoglobin (HbAc1) and the circadian period length of skin fibroblasts derived from type 2 obese and non-obese diabetic patients was observed. Although we have launched several lines of the experiments addressing the molecular mechanisms underlying this intriguing phenomenon, the basis of demonstrated here correlation cannot yet be readily explained by this study. The potential link between T2D and circadian clock machinery in human individuals that we have uncovered opens several paths for further exploring this conjunction and may have important scientific and clinical implications.

Application of circadian bioluminescence recording in the primary cells cultured in vitro for studying human circadian oscillator properties

The application of studying circadian bioluminescence in skin fibroblasts for studying human peripheral oscillator originally proposed by Steven Brown represents unique non-invasive approach for studying human circadian clock in physiology and pathophysiology [132]. The analysis of the clock machinery in humans required heavy and prolonged protocols including sampling procedures under laboratory controlled conditions. For example, standard protocols involve maintaining the volunteer upon non-circadian schedules like “constant routine “ or

“forced desynchrony” and observe his circadian hormones and body temperature cycles [163].

124 Even if this method represents the standard to evaluate the human behavioural period length, it is expensive and highly disturbing (sleep deprivation for several days) and time consuming for the volunteer. Less invasive methods has emerged to study the human clockwork in vivo like the collection of blood and saliva [31,128], or more recently the analysis of breath metabolome [164]. However, the precision of these less invasive methods is limited because of the inter-individual variability. Regarding the difficulty of in vivo methods, in vitro techniques has been developed based on the studies demonstrating that cells can be synchronized in vitro [130] and transduced with a bioluminescent circadian reporter allowing the visualization of oscillation profiles [132]. This approach was used for the first time by Emi Nagoshi to provide compelling evidence that peripheral clocks are cell-autonomous. In addition, clock properties of human skin fibroblasts correlated with rhythmic human behaviour [134], thus validating the method application for studying human clocks by such in vitro approach.

The circadian characteristics did not differ among the groups of cells in our cohort.

This study allowed assessing for the first time the circadian rhythm in skin fibroblasts from healthy subjects, T2D and obese patients. The circadian period lengths were not different among the groups, suggesting no disruption of the circadian rhythm. Cells from T2D patients had similar oscillation characteristics as cells from the healthy subjects, whereas a phase advance was observed in fibroblasts from obese patients.

Considering the small number of patients included in this study and inter-individual variations in the oscillator profile insight each of the groups, the lack of overall significant difference in the oscillator characteristics between the groups might be explained by the small numbers of the subject within each group. To overcome this limitation of our initial study, larger cohort (100 T2D patients and 200 non T2D patients) is under recruitment.

125 At the molecular level, it was demonstrated that the clock gene expressions could vary in mice fed with different diets like high fat diet [63,71,72]. In our protocol, the diet was controlled to ensure that potential effects observed on the circadian rhythm were not related to the diet, but to the pathology. The oscillation profiles of the expression of clock genes and clock-controlled genes showed no differences between the groups possibly because the chronotypes and the period lengths were similar. In line with these results, cells incubated with a serum from another group of patient did not show modifications in their oscillation profiles. This suggests that in our cohort, the effect of the sera was not sufficient to modify the clock properties.

Similarly to our study but in another tissue, no difference was found between the oscillation profiles of core clock genes from WAT biopsies obtained from lean, obese and T2D subjects [165]. In this study from Otway et al, the tissue biopsies were obtained with 6-hour resolution, which is obviously as frequent as an “in vivo” study of this kind could be performed in human beings. Contrary to our study, Tahira et al showed different expression profiles of clock genes in mononuclear cells from 10 obese patients compared to 10 healthy subjects. In their study, the blood from these patients was collected every 3 hours between 9 AM and 9 PM and RNA was extracted from blood mononuclear cells to perform qPCR [78].

These conditions seem ideal and closer to the physiology to compare the expression profiles between two groups but were not realizable in our study since we need to propagate the cells and synchronize them before RNA extraction. It is not excluded that we could point out some differences between the groups by increasing the number of subjects for the analysis of clock gene expressions.

Therefore, measuring circadian oscillations using a bioluminescence reporter assay represents an advantage for this kind of study, and is probably the only approach allowing discovery of physiologically plausible differences in period length due to high temporary resolution.

126 Lentiviral-reporter-based methodologies such as the one employed here permit much greater flexibility and resolution in the study of intrinsic circadian properties than serial sampling methods. Our study was the first that looked at the gene expression profiles in human primary fibroblasts from healthy, T2D and obese patients.

HbA1c levels are inversely correlated with the period lengths in T2D patients.

We analyzed the correlations between the period length and the blood parameters in each group of patients to further look at the impact of serum factors. The inverse correlation we found between HbA1c level and the period length in fibroblasts from T2D patients may suggest that the circadian oscillator is implied in this pathology development. In line with this correlation, the average HbA1c level was higher in blood from not compensated T2D patients having an HbA1c > 7.1 %, whereas the period length was shorter. Two previous studies have demonstrated correlation between the expression of clock genes in human tissues and the HbA1c in T2D subjects. Stamenkovic et al showed that the transcript level of PER3 and CRY2 were inversely correlated with HbA1c level in human islets from T2D donors [113]. In leucocytes from patients with T2D, Ando et al showed that BMAL1, PER1 and PER3 expression levels were inversely correlated with HbA1c values [114].

The observed inverse correlation between HbA1c level and the period length was more pronounced in T2D non-obese patients than in the entire group of T2D patients. This correlation was weaker in the subgroup of obese T2D patients. This could be the effect of the range of the period values that was smaller in cells from T2D obese (23.6-25.1 hours) than from T2D non-obese group (23.2-25.5 hours). Increasing massively the number of subjects would definitively give an answer to this question. An epidemiological study is necessary to integrate all the patient characteristics including for example eating behaviour and medications.

127 The glycemia was inversely correlated to the period length in cells derived from T2D patients and from T2D obese patients but not from T2D non-obese patients. The period length was shorter for fibroblasts from not compensated patients, which corroborates the correlation between glycemia and period length in cells from T2D group. Even if the patients were under controlled-diet, the HbA1c level remains the reference to control the evolution of the diabetes compared to the glycemia that is less reliable.

To conclude, the correlations showed that the period length was shorter for fibroblasts from not compensated patients than from compensated patients. The inverse correlations found between HbA1c and the period length in the group of T2D patients suggests a reciprocal connection between the circadian rhythm and the glucose metabolism.

Unravelling the mechanism of the observed inversed correlation between HbAc and oscillation period in the cells derived from T2D donors

One possible scenario of these correlations between HbA1c and the period length might be that chronic exposure to high levels of glucose in T2D promotes tissue-specific alterations leading to changed properties of the circadian oscillator. For example, two recent studies have demonstrated that modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) might impact on phase and period length of circadian oscillations. In mouse cardiomyocytes, BMAL1 was identified as an O-GlcNAc-modified protein [53]. Moreover, increasing protein O-GlcNAcylation through inhibition of O-GlcNAcase resulted in diminished PER2 protein levels, and phase advance in the SCN clock. In addition, Drosophila melanogaster dPER protein O-GlcNAcylation impacts critically on clock properties [52]. Taken together these recent findings suggest that protein O-GlcNAcylation influences the phase and the period length of the circadian clock in certain models.

128 The absence of different patterns of O-GlcNAcylation of total proteins of fibroblasts from healthy and T2D group at two time points could be explained by the high amount of proteins that are affected by this post-translational event. BMAL1 and PER2 have been showed as O-GlcNAcylated proteins [52,53], and since they are antiphasic, one possible explanation is that the total amount of O-GlcNAcylated proteins is conserved. The inhibition of OGA by PUGNAc did not provide differences of glycosylated proteins. Since phosphorylation and O-GlcNAcylation act in an opposite manner [54], it would be interesting to look whether the inhibition of phosphorylation would have a more important impact on the oscillation of clock proteins compared to an inhibition of OGA by PUGNAc. This would lead to better comprehension of the interplay between the post-translational events occurring on clock proteins and the influence on the modulation of the circadian rhythm.

In general, high number of recent studies demonstrated a direct influence of post-translational or epigenetic modifications of clock genes in pathological situations. For example, it is known that BMAL1 transcription factor is SUMOylated in mouse liver [166]. SUMOylation of CLOCK transcription factor impacts on the transcriptional activity of estrogen receptor α that is directly incriminated in breast cancer development [167]. Recently, it has been shown that Per2 gene could be affected in glioma cells because of the methylation of its promoter, and detecting this epigenetic modification would represent a major progress in the treatment of this cancer [168]. These post-translational and epigenetic modifications are more and more studied in oncology, but it could be assumed that similar alterations are present in other chronic diseases such as diabetes or obesity. The haemoglobin is glycated by an non enzymatic process that need to be explored to partly explain the inverse correlation.

129 The RNA sequencing revealed the influence of the chronotype on gene expression.

In an attempt to unravel the mechanisms underlying the inverse correlation between the HbA1c level and the period length of fibroblasts from T2D patients, the RNA-seq aiming to determine the genes differentially expressed between compensated and not compensated patients has been launched. The differential gene expression implied in glucose metabolism and the influence of the chronotype on the transcript expression could partly explain the inverse correlation we found.

Genes implied in glucose metabolism are differentially expressed among the cells from T2D patients.

It has been found that lower expression of Dio2 is associated with reduction of obesity in mouse model of diet-induced obesity [169]. ICAM1 was more expressed in patients with a short period length than in patients with a longer period length. This gene was particularly interesting since it has been shown to be rhythmically expressed and up-regulated by CLOCK in mouse endothelial cells [170]. In T2D patients, it has been demonstrated that BMI and waist circumference are positively correlated with ICAM1 and that HbA1c correlate with ICAM1 independently of age, gender and BMI [171]. Recently, Kulkami and colleagues found that T2D patients with high insulin resistance measured by HOMA-IR have high

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