Circadian oscillations in primary skin fibroblasts from non-diabetic and diabetic individuals in the presence of their own serum
Consistent with previous reports , we found high-amplitude anti-phasic circadian oscillations of Bmal1-luc and Per2-luc reporters in human primary skin fibroblasts (Fig. 1a). Bmal1-luc oscillatory profiles were recorded from fibroblast cells derived from 17 individuals with type 2 diabetes (eight non-obese and nine obese) and compared with 11 non-diabetic counterparts (Table 1). Recording for each cell line was first conducted in the presence of either 15% FCS or 15% of each individual’s own serum (ESM Fig. 1). While no significant differences in period length were observed within the non-diabetic control group when analysed in the presence of FCS or human serum, the oscillation period was significantly shorter in the type 2 diabetes group (particularly the obese subgroup) in the presence of participants’ own sera (ESM Table 5). We next compared cellular oscillations in the non-diabetic and type 2 diabetes groups in the presence of each individual’s own serum and also performed ‘cross-serum’ experiments using serum from a person of the other subgroup (non-diabetic control or type 2 diabetes, respectively) (ESM Fig. 2a). In this experiment, no significant effects of serum factors from non-diabetic and diabetic individuals on fibroblast rhythm were observed (ESM Fig. 2b).
Therefore, for the rest of the study we conducted the analyses of cellular circadian properties in the presence of 15% of each participant’s own serum, representing the closest approximation to the in vivo situation. Under these conditions, no significant differences in oscillation period length were observed between the fibroblasts derived from skin biopsies from non-diabetic individuals and individuals with type 2 diabetes (Fig. 1b, Table 1). Consistently, comparison of temporal expression profiles of endogenous core-clock components in the fibroblasts derived from non-diabetic and diabetic individuals’ skin biopsies also revealed no significant differences in expression levels of BMAL1, REV-ERBα (also known as NR1D1), DBP and PER1 transcripts (ESM Fig. 3).
HbA1c inversely correlates with skin fibroblast circadian period in individuals with type 2 diabetes
To test the possible association of cellular circadian characteristics with the progression of type 2 diabetes pathophysiology, we calculated the correlations between clinical and metabolic characteristics of the participants and Bmal1-luc period length measured in skin fibroblasts in vitro (Table 2). Strikingly, our analysis revealed a significant inverse correlation between HbA1c blood levels and circadian period length measured in skin fibroblasts in vitro, considering study participants overall (Table 2). When the correlation analysis was performed separately for each study group, this inverse correlation was maintained within the type 2 diabetes group: cells from individuals with type 2 diabetes with highest HbA1c exhibited the shortest oscillation period (Fig. 2, Table 1). Moreover, fibroblasts derived from individuals with poorly controlled type 2 diabetes (HbA1c >54.1 mmol/mol [7.1%]) had a significantly shorter period length than those from individuals with controlled type 2 diabetes (HbA1c ≤54.1 mmol/mol [7.1%]) (23.9 ± 0.17 h vs 24.7 ± 0.25 h respectively, Table 1). This inverse correlation was not observed for the non-diabetic control group (Fig. 2).
The correlation between HbA1c values and cellular period length recorded in vitro was stronger for the subgroup of individuals who were non-obese and had type 2 diabetes (ESM Fig. 4a), consistent with the significant inverse correlation between blood glucose and cellular oscillation period in type 2 diabetes (ESM Fig. 4b). In addition, in the diabetic non-obese group, there was a significant inverse correlation between serum triacylglycerol levels and skin fibroblast period length (ESM Fig. 4c, ESM Table 3). No significant correlation was observed between BMI and cellular period length (Table 2). Finally, age and sex did not have an impact on the cellular oscillatory properties (ESM Figs 5, 6).
RNAseq analysis of human skin fibroblasts from the type 2 diabetes group reveals genes differentially expressed in concordance with diabetes severity and individual chronotype
To obtain mechanistic insights into the inverse correlation between circadian period of the skin fibroblasts and HbA1c values that we observed in individuals with type 2 diabetes, we performed global gene expression profiling. RNAseq analysis was conducted in cells derived from individuals with type 2 diabetes 24 h following in vitro synchronisation. Type 2 diabetes samples were grouped according to cellular period length (Table 3) and differential gene expression analysis was performed by permutation analysis, randomly sampling 100 technical replicates per comparison in order to robustly call differentially expressed genes (ESM Methods). Interestingly, such analysis conducted between cells derived from the participants with type 2 diabetes with either early or late chronotype (as evaluated by MSF_sc) revealed 1048 differentially expressed genes (Table 3). Next, analysis considering cells from type 2 diabetes samples based on an individual’s clinical characteristics revealed several groups of differentially expressed genes involved in cell surface receptor signalling, cell adhesion and additional pathways (ESM Table 6).
Importantly, RNAseq analysis of genes differentially expressed between cells exhibiting a ‘short’ period length (defined by us as ≤24 h) and those with a ‘long’ period length (≥25 h) identified ICAM1, encoding for a cell adhesion molecule (Table 3). Moreover, ICAM1 was differentially expressed depending on BMI (ESM Table 6).
ICAM1 exhibits different temporal patterns in human skin fibroblasts derived from non-diabetic individuals, individuals with well-controlled type 2 diabetes and individuals with poorly controlled type 2 diabetes
ICAM1 gene expression profiling from cells harvested ‘around the clock’ (i.e. at different times of day after circadian rhythm synchronisation) revealed higher expression levels in the fibroblasts derived from individuals with poorly controlled type 2 diabetes (exhibiting HbA1c >54.1 mmol/mol /7.1%), as compared with the cells established from counterparts with well-controlled blood glucose levels (Fig. 3a). This difference was observed at all time points, and it reached statistical significance at circadian time (CT) 20, with CT defining the time after in vitro synchronisation (Fig. 3a). In view of previously reported evidence implicating ICAM1 in type 2 diabetes pathology , we compared its temporal expression between the fibroblasts derived from skin biopsies from non-diabetic control individuals and individuals with type 2 diabetes (Fig. 3b). ICAM1 exhibited very low expression levels in fibroblasts derived from control individuals, and this expression was not qualified as circadian rhythmic according to CosinorJ analysis ). Of note, our previously published circadian transcriptome from human skeletal muscle biopsies derived from non-diabetic individuals revealed robust circadian expression of ICAM1 in vivo . ICAM1 levels increased two- to 3.5-fold in the counterparts established from individuals with type 2 diabetes between CT 24 and CT 32. Overall, ICAM1 expression in cells established from skin biopsies from individuals with type 2 diabetes synchronised in vitro exhibited a circadian profile, with a period length of 26.44 ± 0.32 h according to the CosinorJ algorithm, and the highest expression occurring at CT 24 (Fig. 3b).
The circadian transcription factor CLOCK binds rhythmically to the ICAM1 gene in fibroblasts from individuals with type 2 diabetes and correlates with gene expression
CLOCK has been suggested to induce ICAM1 transcription through its binding to the ICAM1 gene E-box-like enhancer region in cultured mouse hepatocytes . To test whether a similar mechanism may account for differential rhythmic expression of ICAM1 in human fibroblasts, we performed a ChIP assay. This assay demonstrated CLOCK binding to the ICAM1 promoter region in human fibroblasts (Fig. 4a). Temporal analysis of CLOCK binding to ICAM1 promoter in synchronised skin fibroblasts revealed a lower level of binding in fibroblasts derived from non-diabetic control individual biopsies compared with the cells established from diabetic counterparts. This difference was particularly pronounced at CT 20 (Fig. 4b), where the binding of CLOCK reached its maximum in cells derived from individuals with type 2 diabetes, prior to the maximum ICAM1 mRNA expression measured at CT 24 by qRT-PCR (Fig. 3b), suggesting a potential role of CLOCK binding in the upregulation of ICAM1 rhythmic expression in individuals with type 2 diabetes.