The circadian transcription factor ARNTL2 is regulated by weight-loss interventions in human white adipose tissue and inhibits adipogenesis

Obesity is a significant global health concern, associated with premature aging and increased risk of age-related diseases. Lifestyle factors, such as disrupted sleep-wake cycles and high-calorie diets, contribute to obesity by impacting circadian rhythms. Circadian rhythms are crucial for coordinating metabolic processes and cellular functions within a 24-hour cycle. The central circadian clock in the brain (suprachiasmatic nucleus) is entrained by light, while peripheral clocks are influenced by food intake. Obesity is characterized by white adipose tissue (WAT) expansion, resulting from increased adipocyte size (hypertrophy) and/or number (hyperplasia). Adipogenesis, the differentiation of adipose stem/progenitor cells (ASCs) into adipocytes, contributes to WAT expansion. Adipogenic differentiation is triggered by extracellular stimuli, like insulin and IGF-1, activating PI3K/Akt/mTOR and RAS/MAPK pathways, which regulate the expression of key transcription factors such as C/EBPβ and PPARγ. Cellular clocks interact with adipogenic differentiation regulators. The transcription factors ARNTL1 (BMAL1) and ARNTL2 (BMAL2) heterodimerize with CLOCK, binding to E-box elements in clock-controlled genes (ccgs) to initiate transcription. ARNTL1's role in adipogenesis is known, but ARNTL2's function remains unclear. Weight-loss (WL) interventions, such as caloric restriction, can slow aging and may involve peripheral circadian clocks. This study aimed to determine ARNTL2's role in adipogenesis, hypothesizing that it plays a significant role in the regulation of this process and in the response to weight loss.

Existing literature establishes a strong link between circadian rhythm disruption and obesity. Studies have shown that altered sleep patterns and irregular meal timings can significantly influence the development of obesity and metabolic disorders. The role of the circadian clock components in regulating adipogenesis has been investigated, with ARNTL1 (BMAL1) identified as a key regulator. However, the role of its paralog, ARNTL2 (BMAL2), in adipogenesis remained poorly understood, despite evidence suggesting functional overlap and potential differences in their roles. Previous studies in animal models indicate that ARNTL2 can partially compensate for ARNTL1 deficiency, suggesting distinct but potentially overlapping functions. Weight-loss interventions, particularly caloric restriction, have been shown to have beneficial effects on healthspan and lifespan in various animal models and human studies. These interventions impact peripheral circadian clocks, leading to changes in gene expression and metabolic processes. Our previous work identified several weight-loss target genes in human ASCs, implicating ARNTL2 as a potentially important regulator in adipose tissue biology. These genes play crucial roles in maintaining ASC proliferation and differentiation capacity and preventing cellular senescence.

Human ASCs were isolated from subcutaneous WAT samples of normal-weight, obese, and weight-loss donors undergoing elective abdominal surgery. Whole-genome microarray analysis identified ARNTL2 as a weight-loss-regulated gene. A serum-shock approach was used to synchronize circadian rhythms in ASCs, followed by RNA and protein analyses of ARNTL1, ARNTL2, and C/EBPβ at various time points. Adipogenic differentiation was induced using differentiation medium (DM), and the expression of ARNTL1 and ARNTL2 was monitored over time. Pharmacological inhibition of PI3K/Akt/mTOR and MAPK pathways was used to investigate their roles in ARNTL2 regulation. ARNTL2 was overexpressed in ASCs to determine its impact on adipogenesis, assessing the expression of key adipogenic markers (C/EBPβ, PPARγ, FABP4, ADIPOQ) and lipid accumulation. Western blotting was performed to analyze the protein levels of ARNTL1, ARNTL2, C/EBPβ, and other signaling molecules. The effect of ARNTL2 overexpression on PI3K/Akt/mTOR and MAPK signaling was also examined. ARNTL1 protein half-life was measured using cycloheximide chase assays with and without proteasome inhibitors (MG132). Finally, Western blot analysis of whole sWAT samples from normal-weight, obese, and weight-loss donors was performed to assess ARNTL2 and ARNTL1 protein levels in vivo. Statistical analyses included One-way ANOVA, Bonferroni's multiple comparison test, paired and unpaired t-tests, and Cosinor regression analysis.

Microarray analysis revealed that ARNTL2 mRNA expression was significantly downregulated in ASCs from weight-loss donors compared to normal-weight and obese donors. Serum-shock experiments demonstrated that ARNTL2 protein exhibited a clear oscillatory pattern, while ARNTL2 mRNA showed a less prominent rhythmic expression, suggesting post-translational regulation. In contrast to ARNTL2, ARNTL1 mRNA showed a distinct circadian rhythm. Upon stimulation with adipogenic differentiation medium (DM), ARNTL2 protein levels increased rapidly and remained high, while ARNTL1 protein levels remained relatively unchanged. The study revealed that ARNTL2 protein stability was cooperatively maintained by mTOR and MAPK signaling pathways. Inhibition of both pathways simultaneously was required to reduce ARNTL2 protein levels. Conversely, ARNTL2 overexpression led to impaired mTOR and MAPK signaling. ARNTL2 overexpression significantly reduced adipogenic differentiation as evidenced by decreased mRNA and protein levels of C/EBPβ, PPARγ2, FABP4, and ADIPOQ, accompanied by a significant reduction in lipid accumulation. ARNTL2 overexpression also caused a decrease in ARNTL1 protein levels, suggesting ARNTL2-mediated degradation of ARNTL1. This degradation was confirmed to be mediated by the ubiquitin-proteasome pathway. Furthermore, KLF15 mRNA, a pro-adipogenic transcription factor, was significantly reduced in ARNTL2 overexpressing ASCs. Western blot analysis of sWAT samples from normal-weight, obese, and weight-loss donors showed that ARNTL2 protein levels were significantly higher in the weight-loss group compared to the other two groups, while ARNTL1 was increased in both obese and weight-loss groups. Donor age did not significantly affect ARNTL2 protein levels.

This study provides compelling evidence for the antagonistic relationship between ARNTL1 and ARNTL2 in human adipogenesis. The findings that ARNTL2 protein accumulates in ASCs in response to adipogenic stimuli and its anti-adipogenic function highlight its role as an inducible inhibitor of adipogenesis. The observation that ARNTL2 is regulated by mTOR and MAPK signaling, yet in turn inhibits these pathways, suggests a complex feedback mechanism crucial for maintaining ARNTL2 levels during adipogenesis. The weight-loss-induced increase in ARNTL2 protein in sWAT further supports its role as a regulator of WAT homeostasis. The ARNTL2-mediated degradation of ARNTL1 and the downregulation of KLF15 provide potential mechanisms by which ARNTL2 inhibits adipogenesis. These findings align with the known role of ARNTL1 as a positive regulator of adipogenesis. The observed increase in ARNTL2 in response to weight-loss interventions aligns with our previous findings showing that weight loss promotes the expression of inhibitors of PI3K/Akt/mTOR and MAPK pathways in ASCs. This indicates that the induction of ARNTL2 might be a protective mechanism in response to weight loss, preventing excessive adipogenesis and promoting metabolic health.

This study reveals ARNTL2 as a novel weight-loss-regulated inhibitor of adipogenesis in human ASCs. The antagonistic relationship between ARNTL1 and ARNTL2, together with the impact on key signaling pathways and adipogenic markers, underscores the complexity of circadian regulation in adipose tissue. This research has implications for developing new strategies to treat obesity and improve metabolic health by targeting the ARNTL1/ARNTL2 axis. Future studies should investigate the precise molecular mechanisms underlying ARNTL2's inhibitory effect on adipogenesis and explore its therapeutic potential in obesity management.

The study utilized samples from patients undergoing elective surgery, potentially introducing a bias. The serum-shock model, while effective for inducing circadian gene expression, may not perfectly mimic physiological conditions. The in vitro nature of some experiments limits the direct translatability to in vivo situations. Further research is needed to validate the findings in larger and more diverse populations and to explore potential long-term effects of ARNTL2 modulation.