Biliverdin Reductase A Attenuates Hepatic Steatosis by Inhibition of Glycogen Synthase Kinase (GSK) 3β Phosphorylation of Serine 73 of Peroxisome Proliferator-activated Receptor (PPAR) α

Abstract

Non-alcoholic fatty liver disease is the most rapidly growing form of liver disease and if left untreated can result in non-alcoholic steatohepatitis, ultimately resulting in liver cirrhosis and failure. Biliverdin reductase A (BVRA) is a multifunctioning protein primarily responsible for the reduction of biliverdin to bilirubin. Also, BVRA functions as a kinase and transcription factor, regulating several cellular functions. We report here that liver BVRA protects against hepatic steatosis by inhibiting glycogen synthase kinase 3β (GSK3β) by enhancing serine 9 phosphorylation, which inhibits its activity. We show that GSK3β phosphorylates serine 73 (Ser(P)⁷³) of the peroxisome proliferator-activated receptor α (PPARα), which in turn increased ubiquitination and protein turnover, as well as decreased activity. Interestingly, liver-specific BVRA KO mice had increased GSK3β activity and Ser(P)⁷³ of PPARα, which resulted in decreased PPARα protein and activity. Furthermore, the liver-specific BVRA KO mice exhibited increased plasma glucose and insulin levels and decreased glycogen storage, which may be due to the manifestation of hepatic steatosis observed in the mice. These findings reveal a novel BVRA-GSKβ-PPARα axis that regulates hepatic lipid metabolism and may provide unique targets for the treatment of non-alcoholic fatty liver disease.

Keywords: glycogen synthase kinase 3 (GSK-3); heme oxygenase; liver metabolism; nuclear receptor; peroxisome proliferator-activated receptor (PPAR).

FIGURE 1.

Liver-specific knock-out of BVRA in mice increases blood glucose. A, real time PCR of BVRA in the liver of LBVRA KO and flox mice. B, hepatic immunofluorescence staining for BVRA. Scale bars, 75 μm. C, Western blot of BVRA from different tissues. B, brain; H, heart; K, kidney; L, liver; S, spleen. D, hepatic BVR activity. E–I, measurement of body weight (E), lean and fat mass (F), as well as plasma bilirubin (G), glucose (H), and insulin (I) in LBVRA KO and control flox mice. *, p < 0.05 (versus floxed mice; ± S.E.; n = 6–8/group).

FIGURE 2.

BVRA regulates hepatic insulin signaling. A and B, Western blot and densitometry of the insulin receptor precursor and insulin receptor-β (IRβ) in the liver (A) or AKT1/2 and phosphorylated AKT in the liver (B). C, GTT. D, ITT in LBVRA KO and control flox mice. **, p < 0.01 (versus flox mice; ± S.E.; n = 4); ***, p < 0.001 (versus flox mice; ± S.E.; n = 4).

FIGURE 3.

The loss of hepatic BVRA causes lipid accumulation. A, hepatic fat measured by Echo-MRI. B, biochemical measurement of hepatic triglyceride levels. C, Oil Red O staining of liver sections and densitometry. Scale bars, 50 μm. D–F, representative Western blot of hepatic expression of AMPK and phosphorylated AMPK (D), FAS (E), and phosphorylated ACC (F). *, p < 0.05; **, p < 0.01 (versus flox mice; ± S.E.; n = 4).

FIGURE 4.

GSK3β is more active in the LBVRA KO mice and suppresses glycogen storage. A and B, Western blot and densitometry of hepatic total and phosphorylated GSK3β and levels of serine 9 phosphorylated (pS9) GSK3β measured by ELISA (A) and periodic acid Schiff staining for glycogen in liver (B). Scale bars, 50 μm. C, real time PCR measurement of hepatic expression levels of GYS2 mRNA. D, representative Western blot of hepatic GYS2 and serine 641 phosphorylation of GYS. *, p < 0.05; **, p < 0.01 (versus flox mice; ± S.E.; n = 4).

FIGURE 5.

PPARα expression and activity are reduced in LBVRA KO mice. A and B, Western blot and densitometry of hepatic protein levels of CPT1A and PPARα (A), and real time PCR of CPT1A, CYP2J6, and CYP4A12 mRNA (B). C, real time PCR of CD36, G6Pase, and glucokinase mRNA. D, real time PCR of FGF21 mRNA and plasma FGF21 level in LBVRA KO and control flox mice. *, p < 0.05 (versus flox mice; ± S.E.; n = 4).

FIGURE 6.

GSK3β targets PPARα by phosphorylation at serine 73 for degradation. A, graphical representation of serines in the activation factor-1 A/B domains of rat (rPPARα), mouse (mPPARα), and human (hPPARα). PPARα contains five putative GSK3 consensus phosphorylation sites within the A/B and C domains. B, purified bacterially expressed pMAL-PPARα wild-type was incubated with [γ-³²P]ATP in the presence of increasing amounts of activated GSK3β. C, purified bacterially expressed pMAL-PPARα full-length (FL) and domains (A/B, C, D, and E/F) were incubated with [γ-³²P]ATP in the presence (+) or absence (−) of activated GSK3β (top panel). A Coomassie stain of the in vitro kinase assay was performed (bottom panel; representative of three or more experiments). D, Cos-1 cells were transfected with an HA-tagged ubiquitin expression vector, V5-rPPARα, V5-PPARα S73A, and/or pcDNA GSK3β. The cells were co-treated with vehicle or 50 μm WY-14643 and 5 μm MG132 for 4 h. PPARα protein was immunoprecipitated (IP) and analyzed by Western blotting (WB) using anti-V5 and anti-HA antibodies (representative of three experiments). E, PPARα activity at the minimal promoter PPRE-3tk-luc with increasing doses of GSK3β (0, 100, 200, 400, and 600 ng) with empty vector (100 ng) or PPARα (100 ng) in the presence of vehicle (WY −) or WY-14643 (WY +). *, p < 0.05; **, p < 0.01 (versus WY −, 0 GSK3β, PPARα control; ± S.E.; n = 4). PPARα and GSK3β WT and K85A mutant with the minimal promoter PPRE-3tk-luc were transfected in Cos-7 and treated with WY-14643 or vehicle for 24 h. F, PPARα activity at the minimal promoter PPRE-3tk-luc with WT GSK3β or kinase-dead K85A GSK3β in the presences of vehicle (WY −) or WY-14643 (WY +). *, p < 0.05; **, p < 0.01 (versus WY +, − GSK3β, + PPARα; ± S.E.; n = 4).

FIGURE 7.

PPARα serine 73 phosphorylation is increased in LBVRA KO mice. A, PPARα WT, S73A, and S73D mutants with the minimal promoter PPRE-3tk-luc were transfected in Cos-7 and treated with WY-14643 or vehicle for 24 h. B, COS-7 cells were transfected with a FLAG-tagged PPARα construct for 24 h followed by immunostaining with antibodies to Ser(P)⁷³ PPARα-Ab or FLAG or with Ser(P)⁷³ PPARα-Ab plus blocking peptide used to construct the antibody, as well as negative control with only secondary antibodies. Scale bars, 75 μm. C, levels of phosphorylated serine 73 (green), total PPARα (red), and nuclear staining (blue) with DRAQ5 in the liver of LBVRA KO and flox mice. Scale bars, 50 μm. **, p < 0.01 (versus floxed mice; ± S.E.; n = 4).

FIGURE 8.

Diagram of hepatic BVRA signaling to reduce fatty liver and increase glycogen storage. BVRA reduces biliverdin to bilirubin, which binds to and activates the nuclear receptor PPARα for the burning of fat by β-oxidation and storage of glycogen by increasing GYS2. Additionally, BVRA binds directly to AKT to increase phosphorylation, which inhibits GSK3β activity to increase glycogen and decrease lipid storage in the liver. The BVRA-AKT interaction also increases hepatic insulin sensitivity and glucose uptake.