Abstract

Lifestyle changes, characterized by disrupted biological rhythms, particularly increase the risk of obesity and diabetes. Obesity is characterized by chronic low-grade inflammation of adipose tissue caused by immune-metabolic dysfunctions in mature adipocytes and progenitors, activation of tissue-resident immune cells and infiltration of pro-inflammatory immune cells. Adipocytes can produce inflammatory cytokines (TNFa and IL-6), chemokines (CCL2, CCL5) and adipokines (leptin, adiponectin) which regulate both metabolism and inflammation. Disturbance in the production of these molecules have been directly linked to insulin resistance, ultimately leading to type 2 diabetes (T2D) and fatty liver disease. BMAL2 is a transcription factor belonging to the circadian clock, a transcriptional loop driven by light and food that synchronizes metabolic functions with the rhythm of life. The murine Bmal2 gene is involved in type 1 diabetes and inflammatory mechanisms via the transcriptional control of the cytokine IL-21 expression. In humans, decreased expression in white adipose tissue and certain polymorphisms of BMAL2 are associated with metabolic complications of obesity such as T2D. BMAL2 seems to be regulated during weight loss in humans and inhibit adipogenesis. However, the mechanisms of action of BMAL2 in the control of immune-metabolic dysfunctions in obesity and T2D remain unknown to date. Using adipose tissue samples taken during bariatric surgeries, we demonstrated the association between the expression of BMAL2 and TNFa during obesity in human. Then, we used a Bmal2-/- mouse model fed a normal diet or an obesogenic high fat diet (HFD) to investigate the impact of BMAL2 deletion on glucose metabolism, inflammatory and metabolic functions in the liver and epididymal adipose tissue (EpiWAT). Bmal2-/- mice on HFD developed exacerbated weight gain surprisingly associated with a decrease in lipids storage in EpiWAT and development of greater hepatic steatosis compared to controls. These mice also exhibited a higher level of circulating triglycerides and disturbed glucose metabolism with increased glucose intolerance and decreased insulin sensitivity due to insulin resistance in liver and EpiWAT. In addition, EpiWAT of Bmal2-/- mice was particularly inflamed with increased infiltration by CD8+ T cells and increased expression of inflammatory genes (TNFa, IL-6, IL-17). Interestingly, deletion of Bmal2 significantly altered TNFa circadian expression rhythm in EpiWAT of normal diet and HFD fed mice. We isolated mature adipocytes during EpiWAT digestion and adipose progenitors (PDGFRb+ cells) using flow cytometry cell sorting. In obese Bmal2-/- mice, TNFa expression increased compared to control mice in mature adipocytes as well as in PDGFRb+ cells. Furthermore, frequency of PDGFRb+ cells decreased and was associated with decreased proliferation. Progenitor subpopulations lost their specificities to form a single population that is more homogeneous than in control mice. Analysis of gene expression by bulk RNA sequencing in PDGFRb+ progenitors revealed that the absence of BMAL2 in obese mice was associated with: i) increased expression of numerous inflammatory genes, involved in the NFkB pathway controlling TNFa as well as apoptosis markers, ii) decreased expression of some key players in cellular maintenance pathways (ex: TGFb, PI3K- AKT). Taken together, these results suggest that a defect in the expression of BMAL2 promotes a pro-inflammatory phenotype in adipocyte progenitors and deregulates their metabolic functions, yet necessary for their differentiation to support adequate lipid storage in an obesogenic context, and finally promotes hepatic steatosis and metabolic complications of obesity that are involved in the development of T2D.

Keywords: circadian clock, obesity, type 2 diabetes, hepatic steatosis, NASH, inflammation, insulin resistance, adipose tissue, adipocytes, adipose progenitors