Figure 1.
Naringenin induces activation of PPARα and PPARγ ligand-binding domains.
HG5LN reporter cells expressing GAL4-PPARα (a) and GAL4-PPARγ (b) reporters were treated with increasing concentrations of naringenin. Naringenin dose-dependently activated PPARα reaching 24%±0.2% induction at 240 µM (P<0.001); and activated PPARγ up to 57%±0.3% at 80 µM (P<0.005). Data is presented as percent activation relative to 1 µM of classical agonists GW7647 and BRL49653, respectively. (c) LanthaScreen TR-FRET assay, demonstrating that naringenin did not affect the binding of the PGC1α co-activator peptide to recombinant PPARα LBD. (d) In contrast, the classical PPARα agonist GW7647 induces a dose-dependent binding of PGC1α to PPARα in the same assay.
Figure 2.
Naringenin is a partial agonist of LXRα ligand-binding domain.
(a) LXR-alpha-UAS-bla HEK 293T cells were stimulated with 4.7 nM TO901317 and exposed to increasing concentrations of naringenin. Naringenin dose-dependently inhibited LXRα activity, reaching 28.4%±0.4% (p<0.01) and 39.1%±9.4% (p<0.05) at concentrations of 126 µM and 400 µM, respectively. (b-d) Lanthascreen TR-FRET assay, demonstrating that naringenin weakly increased the binding of Trap 220/Drip-2 co-activator peptide to recombinant LXRα LBD, and inhibited this binding in the presence of TO901317, LXRα classical agonist. (b) Naringenin is a weak agonist, enhancing the binding of the LXRα LBD to the Trap 220/Drip-2 co-activator moderately, yet significantly, in a dose-dependent manner reaching 38.0%±2.8% activation. (c) LXRα agonist TO901317 strongly enhanced co-activator binding. (d) When treated with 250 nM TO901317, increasing concentrations of naringenin led to an inhibition of the TR-FRET signal, reaching 15.0%±4.1% inhibition (p<0.01) at 133 µM.
Figure 3.
Naringenin activates PPRE-driven and inhibits LXRE-driven gene expression in human hepatocytes.
(a) Naringenin dose-dependently enhanced PPRE activity, in Huh7 cells transiently transfected with a PPRE reporter, reaching 17%±7% (p<0.05) at 200 µM. Induction was not different from PPAR agonists WY14,643 and ciglitazone. (b) Naringenin induced the expression of PPARα coactivator PGC1α by 14-fold (p = 0.001) as well as PPARα-regulated fatty acid oxidation genes CYP4A11/22, ACOX, UCP1 and ApoAI. Huh7 cells were treated with naringenin for 24 hours and mRNA isolated and anlysed by qRT-PCR. (c) Naringenin dose-dependently suppressed LXRE activity, in Huh7 cells transiently transfected with a LXRE reporter, reaching a 50.3%±2.6% (p<0.001) inhibition at 150 µM. (d) Naringenin inhibited the expression of LXRα-regulated lipogenesis genes ABCA1, ABCG1, HMGR, and FASN. Cell viability under all conidtions was greater than 95%.
Figure 4.
Naringenin induced a fasted-like state in hepatic lipid metabolism.
(a) Huh7 cells were stimulated for 24 hours with 200 µM naringenin, 10 µM WY14,643, or 10 µM ciglitazone. Naringenin treatment led to a 73%±9% (p<0.001) reduction in ApoB production, while WY14,643 led to a 33%±12% (p<0.01) reduction. Treatment with cigilitazone did not lead to a significant change in VLDL production. (b) Primary rat hepatocytes were stimulated with 200 µM naringenin or 10 µM WY14,643. Naringenin treatment led to a 61% (p<0.001) reduction in triglyceride production and 17% increase in ketone body formation, not different from WY14,643. However, naringenin treatment led to a 32%±11% (p = 0.005) reduction in bile salt production, while WY14,643 did not. Urea accumulation in the media did not change significantly. (c) Intracellular levels of triglycerides in primary rat hepatocytes stimulated with naringenin. A slight decrease is observed. (d) Naringenin effect on SRE-driven gene expression. We show that naringein induces LDLR transcription by 26% (p = 0.02) while inhibiting HMGCS transcription by 13% (p = 0.001). It is thought that each promoter is regulated by a different SREBP isoform.
Table 1.
Real-Time qRT-PCR Primers.