No abstract

Boosting energy expenditure by harnessing the activity of brown adipocytes is a promising strategy for combatting the global epidemic of obesity. Many studies have revealed that the β 3 -adrenergic receptor agonist is a potent activator of brown adipocytes, even in humans, and PKA and p38 MAPK have been demonstrated for regulating the transcription of a wide range of critical genes such as Ucp1 . We previously revealed that the PKA-ASK1-p38 axis is activated in immature brown adipocytes and contributes to functional maturation. However, the downstream mechanisms of PKA that initiate the p38 MAPK cascade are still mostly unknown in mature brown adipocytes. Here, we identified the ASK family as a crucial signaling molecule bridging PKA and MAPK in mature brown adipocytes. Mechanistically, the phosphorylation of ASK1 at threonine 99 and serine 993 is critical in PKA-dependent ASK1 activation. Additionally, PKA also activates ASK2, which contributes to MAPK regulation. These lines of evidence provide new details for tailoring a βAR-dependent brown adipocyte activation strategy.

21Boosting energy expenditure by harnessing the activity of brown adipocytes is a promising 22 strategy for combatting the global epidemic of obesity. Many studies have revealed that the β3-adrenergic 23 receptor agonist is a potent activator of brown adipocytes, even in humans, and PKA and p38 MAPK have 24 been demonstrated for regulating the transcription of a wide range of critical genes such as Ucp1. We 25 previously revealed that the PKA-ASK1-p38 axis is activated in immature brown adipocytes and 26 contributes to functional maturation. However, the downstream mechanisms of PKA that initiate the p38 27 MAPK cascade are still mostly unknown in mature brown adipocytes. Here, we identified the ASK family 28 as a crucial signaling molecule bridging PKA and MAPK in mature brown adipocytes. Mechanistically, the 29 phosphorylation of ASK1 at threonine 99 and serine 993 is critical in PKA-dependent ASK1 activation. 30Additionally, PKA also activates ASK2, which contributes to MAPK regulation. These lines of evidence 31 provide new details for tailoring a βAR-dependent brown adipocyte activation strategy. 32 and the approved dose exhibits only limited effects; hence, off-target effects of β3AR agonists and cross-57 activation of β1AR and β2AR should be considered. Clinically, constructing selective methods for activating 58 β3AR on brown and/or beige adipocytes is crucial, but it is of critical importance to understand the signaling 59 mechanisms induced by β3AR agonists in mature brown adipocytes to fully understand the potential side 60 effects of β3AR agonism. However, a limited number of studies have uncovered the involvement of several 61 kinases in β3AR signaling [16][17][18][19]; specifically, the downstream targets of PKA are not fully known. 62Among a wide variety of signaling pathways, the stress-responsive mitogen-activated protein 63 kinase (MAPK) pathway regulates multiple functions such as gene expression and cell death depending on 64 cell types [20,21]. Canonically, three-tiered cascades, MAPK kinase kinase (MAP3K)-MAPK kinase 65 (MAP2K)-MAPK, are the central components of the MAPK pathway, and a tremendous variety of 66 signaling is converged on two exclusive MAPKs, namely, p38s and JNKs [21]. The apoptosis signal- 67regulating kinase (ASK) family, which consists of ASK1, ASK2, and ASK3, is a member of MAP3K and 68 shares similar amino acid sequences throughout the kinase domain but functions differently in many cases 69 [22][23][24]. Additionally, ASKs can form heteromeric complexes and are mutually interactive in some cases 70 [25][26][27][28]. Although the key role of ASK1 is cell death regulation [29][30][31][32][33], we previously reported that ASK1 71 also functions as a differentiation regulator through gene induction in immature brown adipocytes and 72 contributes to functional maturation [9]. However, there have been no reports regarding the functions of 73 the ASK family in mature brown adipocytes. Importantly, cell signaling patterns are incredibly diverse and 74 depend on many parameters,...