Bilirubin Binding to PPARα Inhibits Lipid Accumulation

Abstract

Numerous clinical and population studies have demonstrated that increased serum bilirubin levels protect against cardiovascular and metabolic diseases such as obesity and diabetes. Bilirubin is a potent antioxidant, and the beneficial actions of moderate increases in plasma bilirubin have been thought to be due to the antioxidant effects of this bile pigment. In the present study, we found that bilirubin has a new function as a ligand for PPARα. We show that bilirubin can bind directly to PPARα and increase transcriptional activity. When we compared biliverdin, the precursor to bilirubin, on PPARα transcriptional activation to known PPARα ligands, WY 14,643 and fenofibrate, it showed that fenofibrate and biliverdin have similar activation properties. Treatment of 3T3-L1 adipocytes with biliverdin suppressed lipid accumulation and upregulated PPARα target genes. We treated wild-type and PPARα KO mice on a high fat diet with fenofibrate or bilirubin for seven days and found that both signal through PPARα dependent mechanisms. Furthermore, the effect of bilirubin on lowering glucose and reducing body fat percentage was blunted in PPARα KO mice. These data demonstrate a new function for bilirubin as an agonist of PPARα, which mediates the protection from adiposity afforded by moderate increases in bilirubin.

Fig 1. Structural of PPARα ligands.

(A) Comparison of structures of WY 14, 643, fenofibrate and bilirubin. (B) Arachidonic acid is the precursor for CYP epoxygenase (2C and 2J) production of 5,6-, 8,9-, 11,12-, and 14, 15- epoxyeicosatrienoic acids (EETs).

Fig 2. Bilirubin binds to the ligand-binding pocket of PPARα.

(A) Bilirubin docked into PPARα binding pocket. (B) Bilirubin binds in the same site occupied by the known PPARα ligand GW735 [19]. Bilirubin and the ligand are depicted in green and magenta carbon skeleton, respectively.

Fig 3. Bilirubin and biliverdin activate PPARα activity.

To determine if bilirubin or biliverdin activate PPARα activity we used Cos7 cells that were transiently transfected with a minimal PPARα responsive promoter luciferase construct (PPRE-3tk-luc) for 24 hours along with empty vector and vector containing PPARα cDNA (overexpression). We treated for 24 hours with a dose dependent increase of biliverdin (BV) (A) or bilirubin (BR) (B). *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus 0 μM PPARα); (±S.E.; n = 4). (C) To compare biliverdin (BV), WY 14,643 (WY), and fenofibrate (Feno) on PPARα activity, we use the minimal promoter PPRE-3tk-luc luciferase construct and treated for 24 hours with PPARα overexpressed and then treated with 50 μM each for 24 hours. ****, p < 0.0001 (versus 0 μM Veh); $ and ^^, p < 0.001 (versus 0 μM BV and Feno, respectively); (±S.E.; n = 4).

Fig 4. Bilirubin binds directly to PPARα to increase endogenous gene activity.

(A) Western of PPARα and HSP90 in lentiviral overexpression of PPARα and vector in 3T3-L1 cells. (B) Bilirubin or WY 14,643 linked sepharose resins were used to determine direct binding to PPARα. (C) Bilirubin or biliverdin linked sepharose resins were used to determine direct binding to PPARα. (D) The PPARα overexpression and vector 3T3-L1 cells were treated for 24 hours with biliverdin (BV) (50 μM), WY 14,643 (WY) (50 μM), or fenofibrate (Feno) (50 μM). RNA was extracted and CD36, CPT1, and FGF21 expression was measured by Real-time PCR. ***, p < 0.001 (versus veh 3T3-Vector); ^, p < 0.05 (versus veh 3T3-PPARα); ^^, p < 0.01 (versus veh 3T3-PPARα); ^^^, p < 0.001 (versus veh 3T3-PPARα); $, p < 0.05 (versus WY 3T3-PPARα); $ $, p < 0.01 (versus WY 3T3-PPARα); #, p < 0.05 (versus BV 3T3-PPARα); (±S.E.; n = 3). (E) The mouse hepa1c1c7 liver cells overexpressing PPARα were treated in dialyzed FBS for 24 hours with biliverdin (BV) (50 μM), WY 14,643 (WY) (50 μM), or fenofibrate (Feno) (50 μM). RNA was extracted and mRNA expression was measured by Real-time PCR. ^, p < 0.05, ^^, p < 0.01, and ^^^, p < 0.001 (versus veh 3T3-PPARα); $, p < 0.05, $ $, p < 0.01, $ $ $, p < 0.001, $ $ $ $, p < 0.0001 (versus WY 3T3-PPARα); ###, p < 0.001, #, p < 0.01, ####, p < 0.0001 (versus BV 3T3-PPARα); (±S.E.; n = 3).

Fig 5. Biliverdin reduces lipid accumulation more than other PPARα ligands.

(A) Lipid accumulation was measured by nile red staining (green) and densitometry in 3T3-L1 cells that were differentiated into mature adipocytes treated with vehicle (Ctrl), biliverdin (10 μM), WY 14,643 (10 μM), or fenofibrate (10 μM) over the 9 day protocol and Real-time PCR analysis of PPARγ2, C/EBPα, FAS, and CPT1. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (versus Ctrl); ^^, p < 0.05 (versus 10 μM WY); $, p < 0.05 (versus 10 μM feno) (±S.E.; n = 3). (B) Lipid accumulation was measured in 3T3-L1 cells that were differentiated into mature adipocytes treated with vehicle (Ctrl), biliverdin (50 μM), WY 14,643 (50 μM), or fenofibrate (50 μM) over the 9 day protocol and Real-time PCR analysis of PPARγ2, C/EBPα, FAS, and CPT1. (versus Ctrl) **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; (versus 50 μM WY) #, p < 0.05; ##, p < 0.001; (versus 50 μM feno) $, p < 0.001 (±S.E.; n = 3).

Fig 6. Bilirubin reduces body weight and body fat percentage.

WT and PPARα KO mice were on a high fat diet for 6 weeks and treated with fenofibrate (FF) or bilirubin (BR) for seven days and body weight (A), percent body fat (B), and lean mass (C) were measured. a, p < 0.05 (KO versus WT Ctrl); b, p < 0.05 (WT FF or BR treated versus WT Ctrl) (±S.E.; n = 5).

Fig 7. The glucose lowering affect of bilirubin is blunted in PPARα KO mice.

WT and PPARα KO mice were on a high fat diet for 6 weeks and treated with fenofibrate (FF) or bilirubin (BR) for seven days and blood glucose (A), plasma insulin (B), alanine aminotransferase (ALT) (C), aspartate aminotransferase (AST) (D), and fibroblast growth factor (FGF21) mRNA in liver (E) and serum levels (F) were measured. a, p < 0.05 (KO versus WT Ctrl); b, p < 0.05 (WT FF or BR treated versus WT Ctrl); c, p < 0.05 (WT BR treated versus WT FF treated); d, p < 0.05 (KO FF treated versus WT FF); e, p < 0.05 (KO BR treated versus WT BR) (±S.E.; n = 5).