Since KYNA concentrations are increased by aerobic exercise

Since KYNA concentrations are increased by aerobic exercise training, we have investigated if it plays a role in peripheral tissue metabolism. Here we show that KYNA regulates adipose tissue energy homeostasis through activation of Gpr35. Activation of this network stimulates the expression of lipid metabolism, thermogenic, and anti-inflammatory genes in the adipose tissue. This suppresses weight gain in animals fed a high-fat diet (HFD) and improves glucose tolerance and adipose tissue inflammation. KYNA/Gpr35 signals through a 2-fold mechanism: (1) activation of Ca2+, ERK, and CREB signaling; stabilization of Pgc-1α1; and induction of downstream genes, and (2) increase of Rgs14 gene expression, a protein G regulatory subunit that mediates a negative feedback loop by interacting with Gαi subunits, thus leading to enhanced β-adrenergic receptor (β-AR) signaling. Importantly, these effects are lost upon genetic deletion of Gpr35, which causes progressive weight gain and glucose intolerance. In addition, exercise-induced adipose tissue browning is compromised in Gpr35 knockout animals. Together, our data uncover a novel muscle to adipose tissue and immune system crosstalk mediated by KYNA and Gpr35, with impact on systemic energy expenditure and inflammation.
Results
Discussion Although KYNA has been reported to accumulate in peripheral tissues in the nM to μM range (Badawy and Bano, 2016, Forrest et al., 2002, Olenchock et al., 2016), its biological activities in those tissues are less well known. To mimic a post-exercise situation, we used a single daily dose of KYNA, which elevates its plasma levels to what we and others have previously reported in exercised mice and humans (Agudelo et al., 2014, Lewis et al., 2010, Schlittler et al., 2016). This resulted in changes in the adipose tissue that increase systemic energy expenditure. Importantly, these changes proved to be strictly dependent on Gpr35 as genetic deletion of this GPCR renders KO mice refractory to the metabolic effects of KYNA. These effects seem to be selective to the subcutaneous and visceral compartments, and to have only minimal effects on classical BAT. This selectivity has been observed before for other mediators of skeletal muscle to adipose tissue communication induced by exercise training such as Irisin (Boström et al., 2012), Meteorin-like (Rao et al., 2014), β-aminoisobutyric aa dutp (Roberts et al., 2014), Il6 (Knudsen et al., 2014), and lactate (Carrière et al., 2014). The immediate KYNA/Gpr35 actions are mediated by intracellular Ca2+ release, ERK1/2 phosphorylation, and Pgc-1α1 activation, a well-established pathway in the regulation of adipocyte energy expenditure (Lindquist et al., 2000, Puigserver et al., 1998, Wang et al., 2013, Zemel et al., 2000). Indeed, the adipocyte gene expression signature we observe downstream of KYNA signaling includes several known players in adipose tissue beiging. This suggests that the adipocyte-autonomous effects of KYNA are dependent on molecules such as Pgc-1α and PRDM16, which have been shown to have essential roles in adipose tissue beiging (Cohen et al., 2014, Kleiner et al., 2012). Interestingly, we observed that KYNA enhances the antagonist effect of propranolol on adipocyte β-ARs, but only in the presence of Gpr35. This is in agreement with the fact that Gpr35 has been shown to signal through Gαq/11/Ca2+ and Gαi/0, which stimulates ERK1/2 but inhibits cAMP signaling (Mackenzie et al., 2011). However, chronic treatment of adipocytes with KYNA elevates Rgs14 levels, which alleviates the dampening effect of Gpr35 on β-ARs. Rgs14 is a structurally and functionally unique member of the RGS family of proteins as it contains RGS, Ras/Rap-binding, and GPR/GoLoco domains (Berman et al., 1996, Cho et al., 2000). The RGS and GPR domains mediate inhibitory interactions with activated and inactive Gα proteins, thus temporally limiting G protein signaling. This crosstalk might have important therapeutic implications as it offers an alternative way to regulate adipocyte β-AR activity and energy expenditure by combining Gpr35 and β-AR agonists at lower doses (thus reducing unwanted systemic effects). We could demonstrate this in vitro and in vivo by combining KYNA with a low dose of isoproterenol. Of the three adipose depots we analyzed, iWAT expresses the highest Rgs14 levels (47-fold versus liver), followed by eWAT and BAT. This correlates with the levels of Gpr35 expression and with the magnitude of KYNA effects we observe. Notably, a Drosophila melanogaster mutant with reduced expression of Loco (the fly homolog for Rgs14) shows increased fat content and reduced cAMP signaling (Lin et al., 2011). In addition, it has been previously shown that browning of the adipose tissue can be induced even in the absence of β-ARs (de Jong et al., 2017, Razzoli et al., 2015, Ye et al., 2013), which opens interesting windows of opportunity for the use of Gpr35 agonists as browning agents.