The clock is ticking: A role for inflammatory genes, RIPK1 and/or MLKL, in altering circadian clock in obesity?

Obesity is a major public health burden worldwide, where 2/3 Australians are overweight and/or obese. Obesity greatly increases the risk of diabetes, cardiovascular diseases, and cancer, and is characterized by adipose tissue inflammation and insulin resistance. Adipose tissue is composed of adipocytes, along with anti-inflammatory immune cells (e.g., iNKTs). In obesity, the adipose tissue becomes inflamed, recruiting pro-inflammatory immune cells (e.g., macrophages) that further propagate the inflammatory milieu. RIPK1 (Receptor-Interacting serine/threonine Protein Kinase 1) is a central regulator of inflammatory cell function that coordinates inflammation and cell death. Necroptosis is a proinflammatory cell death pathway regulated by RIPK1, RIPK3 and MLKL, which ultimately results in cell membrane rupture. Recently, we showed that silencing RIPK1 in a mouse model of diet-induced obesity reduced fat mass, body weight and adipose tissue inflammation (Karunakaran et al, Nature Metabolism, 2020). Of note, we discovered several novel RIPK1 SNPs (single nucleotide polymorphisms) that correlate with increased genetic risk of obesity. Interestingly, one of the RIPK1 obesity genetic risk variants promotes RIPK1 expression by disrupting the binding of the circadian transcriptional repressor, E4BP4. Our preliminary data reveal that RIPK1 expression is cyclic in normal mice and amplified in obese mice, likely propagating RIPK1-driven inflammation. Further, high calorie diet-induced NF-kB inflammation inhibits repressors of the circadian clock altering behavior to promote obesity. In this project, we will investigate how RIPK1 and MLKL orchestrates adipocyte or macrophage inflammation and cell death, and how this in turn potentially disrupts the circadian clock rhythmicity in vitro. Together with ongoing in vivo studies, these studies will shed light on the mechanisms by which RIPK1 and/or MLKL regulate adipose tissue circadian clock rhythms and hyper-inflammatory responses. Skills, Techniques & Prerequisites: Aseptic cell culture, siRNA transfections, western blotting, ELISAs, qPCR and obesity mouse models. Individuals who are enthusiastic, and willing to work conscientiously to assist with 24h time course studies to deliver project goals are encouraged to apply.