Mirabegron activates energy expenditure in human induced pluripotent stem cell (hiPSC)-derived beige adipocytes via a self-driven loop of acetylcholine-acetylcholine receptor

Abstract

Obesity is a pathological condition of excessive adiposity in the body and is a strong and independent risk factor for a long list of non-communicable diseases and enhances all-cause mortality. Over the last decade, multiple studies have proved that activating the thermogenesis in adipose tissue shows the potential to increase whole-body energy expenditure and ameliorate glucose intolerance. β3 adrenergic receptor (β3-AR)- UCP1 axis is a classic signalling pathway to activate thermogenic cells in adipose tissue, including brown and beige adipocytes. Mirabegron is an FDA-approved drug for overactive bladder (OAB) by selectively evoking β3-AR. Several small-scale clinical trials collaboratively supported the efficacy of Mirabegron in enhancing the body’s metabolic rate and explored the appropriate dosage without adverse effects on the cardiovascular system. However, the detailed mechanism of action of Mirabegron remains unclear, especially in beige adipocytes. Firstly, I utilized human-induced pluripotent stem cells (hiPSCs) to generate human beige adipocytes through mesodermal induction, adipocyte progenitor differentiation and beige adipocyte maturation. As a result, the multilocular lipid droplets accumulated in the cells, in compliance with the morphological characters of human beige adipocytes. Time –series RNA alternations consist of three stages in the differentiation process. Particularly, the beige-specific markers at mRNA and protein levels were remarkably induced in the final phase. In addition, the hiPSCs-derived beige adipocytes are functional, supported by fully responsive to several stimuli including insulin, triiodothyronine (T3), noradrenaline (NE) and CL316,243. To explore the effects of Mirabegron on human beige adipocytes, I performed the study of glucose uptake and oxygen consumption rate, showing that Mirabegron induced glucose uptake at a comparable level with insulin. Furthermore, mitochondrial respiration was elevated in a concentration- and time-dependent manner in response to Mirabegron. The mitochondrial stress test showed the enhancement of oxygen consumption rate was caused by both coupled and uncoupled rates. More importantly, pharmacological inhibition of β3-AR completely abolished this increase, indicating the action of Mirabegron is β3-AR dependent. In an effort to clarify the underpinning molecular mechanism, RNA-Sequencing (RNA- Seq) and ATAC-Sequencing (ATAC- Seq) were performed. Based on the RNA-Seq data, the transcription of CHAT and nAChR (CHRNB4 and CHRNA3) was unexpectedly elevated, forming an autonomous cycle involving acetylcholine and nicotinic acetylcholine receptor (nAChR). Instead, the classic thermogenic effector UCP1 was barely augmented by Mirabegron in human beige adipocytes. Additionally, the ATAC-Seq analysis suggested that the enhancement of CHAT expression was likely contributed by CREB phosphorylation instead of chromatin remodelling. To conclude, our hiPSCs-derived beige adipocytes showed properties typical of beige adipocytes in morphology and function. Mirabegron enhanced the energy dissipation by CHAT-nAChR signalling. This work shed new insight into the application of Mirabegron for clinical purposes for obesity and metabolic disease and provides a new strategy to initiate thermogenesis in human beige adipocytes without cardiovascular side effects.