Ongoing work at developing ligands and

Ongoing work at developing ligands and modulators of PPARγ is focused on harnessing its anti-inflammatory properties. In recent years, some PPARγ agonists with anti–inflammatory effects (pioglitazone, Sitagliptin metformin/rosiglitazone combination) have already completed clinical trials. However, even with such promising therapeutic activity, the side effects of thiazolidinedione (TZD) drugs include cardiovascular failure, liver toxicity, bone fractures and potential carcinogenesis, these have greatly limited their clinical use (Lehrke and Lazar, 2005). Therefore, much attention has recently been paid to further optimization of the PPARγ ligands\' structures to decrease or abrogate their side effects. In particular, exploration of natural compounds represents one promising strategy for developing new, safer ligands or modulators of PPARγ (Doshi et al., 2010). Apigenin (Api, 4,5,7-trihydroxyflavone) is a naturally occurring plant flavonoid abundant in various fruits and vegetables (Havsteen, 2002). It has lately gained attention as a beneficial and healthy BQ-788 sodium salt because of its various biological effects and low intrinsic toxicity. Moreover, Api has been demonstrated to possess distinct anti-inflammatory activity in chronic inflammation (Choi et al., 2014) and skin inflammation (Byun et al., 2013). Api is also an inhibitor of NAD+ase CD38 and improves metabolic syndrome (Escande et al., 2013). In addition, Nicholas et al. has found that Api can specifically modulate NF-kB in macrophages by suppression the phosphorylation of p65 (Nicholas et al., 2007). It is noteworthy that Api might be a ligand of PPARγ via structure-based virtual screening (Salam et al., 2008a, Mueller et al., 2008). Hence, further study of Api and the underlying mechanisms related to the PPARγ pathway has potential therapeutic implications. In the current study, we identify Api as an effective ligand of PPARγ in macrophages. Importantly, Api can significantly attenuate obesity-related inflammation and metabolic disorders in high-fat diet-induced mice and ob/ob mice. Furthermore, unlike Rosi, a full ligand-type agonist of PPARγ, Api does not exhibit some adverse effects, such as obvious weight gain, osteoporosis, the increase of small adipocytes in white adipose tissue (WAT) and the accumulation of triglycerides in the serum of obese mice.
Materials and Methods Results
Results
Discussion PPARγ is an attractive pharmacological target for the development of drugs to treat metabolic disorders such as insulin resistance (Xu et al., 2003a), type II diabetes (Saltiel and Olefsky, 1996) and chronic inflammation (Buckingham, 2005). As potent full agonists of PPARγ, thiazolidinediones (TZDs), as a class of antidiabetic drugs that includes Rosi and pioglitazone (Marciano et al., 2014), have been widely used for treating the above mentioned disorders. However, side effects of TZDs have greatly limited their therapeutic use. Although not yet unequivocally demonstrated, it is likely that most side effects of TZDs are associated with their high binding affinities for PPARγ and the resultant over-activation of the classical PPARγ pathway. The fact that ligand binding induced conformational changes in PPARγ offers the opportunity for specific ligand-selective regulation of PPARγ transcriptional activity (Higgins and Depaoli, 2010), prompting researchers to seek and discover some effective modulators of PPARγ, and giving rise to the concept of selective PPAR modulators (SPPARMs). Identifying efficient SPPARMs, which can partially activate PPARγ but without the severe side effects of full PPARγ agonists (Higgins and Mantzoros, 2008, Gregoire et al., 2009), is a promising approach to development of new and safer drugs. Here, we report that Api, a natural PPARγ ligand, can significantly curb obesity-related inflammation, as well as markedly ameliorate the metabolic abnormalities and insulin resistance induced by obesity. Api treatment does not exhibit some of the severe adverse effects of Rosi. Our results suggest that Api may act as a potential SPPARM of PPARγ that is useful for treating metabolic diseases. At the transcriptional level, Api only shows moderate PPARγ transactivation activity, with approximately 50% of the Rosi-induced effect, suggesting that Api partially activates PPARγ. Liang et al. have utilized in vitro studies to show that Api is an allosteric effector of PPARγ and is able to bind to PPARγ (Guevara et al., 1998). Another study by Salam et al. using the induced-fit docking method suggests that Api induces PPARγ conformational change and that the binding of Api to PPARγ is different from Rosi (Salam et al., 2008a). In agreement with these studies, our results confirm that Api binds to PPARγ. Furthermore, we found that both the hinge and LBD domain of PPARγ are important for Api binding, which are different from the domains bound by Rosi. By performing mutation experiments, we have identified the four amino acids (K263, K265, L340 and S342) of PPARγ-located at the hinge (181–289aa) and LBD (289–475aa) domains respectively—necessary for Api binding. Additionally, our auto-docking assay indicates that these four amino acids interact with Api through hydrogen bonds and form a PPARγ-binding pocket. Different from Api, Rosi only binds to the LBD domain of PPARγ (Willson et al., 2001), and such a difference in binding may explain the differences in activity between Api and Rosi. Importantly, our in vivo experiments demonstrate that Api improved the glucose tolerance and insulin resistance induced by a high-fat diet without any adverse effects, including the weight gain, hepatic lipid accumulation and osteoporosis seen with Rosi. Herein, we propose that Api is a promising SPPARM of PPARγ.