Pyrvinium Treatment Confers Hepatic Metabolic Benefits via β-Catenin Downregulation and AMPK Activation

Abstract

Genetic evidence has indicated that β-catenin plays a vital role in glucose and lipid metabolism. Here, we investigated whether pyrvinium, an anthelmintic agent previously reported as a down-regulator of cellular β-catenin levels, conferred any metabolic advantages in treatment of metabolic disorders. Glucose production and lipid accumulation were analyzed to assess metabolic response to pyrvinium in hepatocytes. The expression of key proteins and genes were assessed by immunoblotting and RT-PCR. The in vivo efficacy of pyrvinium against metabolic disorders was evaluated in the mice fed with a high fat diet (HFD). We found that pyrvinium inhibited glucose production and reduced lipogenesis by decreasing the expression of key genes in hepatocytes, which were partially elicited by the downregulation of β-catenin through AXIN stabilization. Interestingly, the AMPK pathway also played a role in the action of pyrvinium, dependent on AXIN stabilization but independent of β-catenin downregulation. In HFD-fed mice, pyrvinium treatment led to improvement in glucose tolerance, fatty liver disorder, and serum cholesterol levels along with a reduced body weight gain. Our results show that small molecule stabilization of AXIN using pyrvinium may lead to improved glucose and lipid metabolism, via β-catenin downregulation and AMPK activation.

Keywords: AMPK activation; beta catenin; gluconeogenesis; lipid metabolism; pyrvinium.

Figure 1

Pyrvinium decreased gluconeogenesis and lipogenesis in hepatocytes. (A) Inhibition of pyruvate-induced glucose production in primary mouse hepatocytes by pyrvinium. Cells were treated with pyrvinium for 24 h, then fasted for 4 h prior to glucose production assay. (B) Inhibition of gluconeogenic gene expression in mouse primary hepatocytes incubated with pyrvinium 100 nM for 24 h. (C) Relative mRNA expression of gluconeogenic genes PEPCK and G6Pase in Huh7 cells treated with pyrvinium for 24 h in the medium containing 1g/L glucose. (D) Representative images of Nile red staining for lipids in Huh7 cells. The cells were treated with indicated concentrations of pyrvinium for 36 h and then together with 200 μM oleate for 16 h. The scale bar is 275μm (E) Relative mRNA expression of lipogenic genes ACAT2, ACACA, and FASN in Huh7 cells treated with pyrvinium for 24 h. * p < 0.05; ** p < 0.01. Data are representative of at least three independent studies.

Figure 2

Role of β-catenin downregulation in the effects of pyrvinium on gluconeogenesis and lipogenesis. (A) Protein expression of AXIN1 and β-catenin in Huh7 cells treated with the indicated concentrations of pyrvinium for 24 h. (B) Dual luciferase reporter assay showing the effects by pyrvinium on Wnt signaling pathway activity in Huh7 cells co-treated with LiCl (GSK3β inhibitor) or WNT3a-conditioned medium. The cells were transfected with TCF/LEF reporter plasmids for 16 h and treated with pyrvinium along with LiCl or WNT3a for additional 24 h. (C) Protein expression of β-catenin in Huh7 cells treated with 100 nM pyrvinium in the presence or absence of LiCl (25 mM) for 24 h. (D) Relative mRNA expression in Huh7 cells transfected with wild type β-catenin, or small interfering RNA (siRNA) against AXIN1 and CSNK1A1; transfection was carried out with 500 ng plasmids or 5 pmol siRNA for 48 h. (E) Protein levels of β-catenin in Huh7 cells transfected with siRNA AXIN1 and CSNK1A1. (F–J) Relative mRNA expression of gluconeogenic genes G6Pase (F), PEPCK (G) and lipogenic genes ACAT2 (H), ACACA (I), and FASN (J) in Huh7 cells with or without β-catenin overexpression, knockdown of AXIN1 and CSNK1A1, respectively, followed by treatment with 50 nM pyrvinium for 24 h. Refer to Figure 2 for cell culture conditions. * p < 0.05; ** p < 0.01, *** p < 0.001. Data are representative of at least three independent studies.

Figure 3

Comparison of pyrvinium effects on gene expression between Huh7 and HepG2 cells. (A–E) Relative mRNA expression of gluconeogenic genes G6Pase (A), PEPCK (B) and lipogenic genes ACAT2 (C), ACACA (D), and FASN (E) in HepG2 and Huh7 cells treated with 50 nM pyrvinium for 48 h in the medium containing 1g/L glucose (A,B) or in normal medium containing 200 μM oleate (C–E). (F) Differential protein expression of β-catenin forms in Huh7 and HepG2 cells. (G) Relative mRNA expression of Wnt target gene CCND1 (encoding Cyclin D1) and (H) β-catenin/FOXO1 target gene IGFBP1 in Huh7 and HepG2 cells treated with pyrvinium100 nM for 24 h. (I) Relative mRNA expression of gluconeogenic and lipogenic genes and Wnt target gene CCND 1 in Huh7 cells treated with 5 μM ICG-001 for 24 h. * p < 0.05; ** p < 0.01. Data are representative of at least three independent experiments.

Figure 4

Role of AMPK activation in the effects of pyrvinium on gluconeogenesis and lipogenesis in Huh7 cells. (A) Effect of AXIN1 overexpression on AMPK phosphorylation in Huh7 cells. The cells were transiently overexpressed with the p3 plasmid containing AXIN1 gene. (B) Effect of pyrvinium treatment on AMPK phosphorylation in Huh7 cells. The cells were treated with varying concentrations of pyrvinium for 24 h. (C) The gene expression of PRKAA1 (encoding AMPK alpha 1) in Huh7 cells transfected with siRNA against PRKAA1. (D) Protein expression of phosphorylated and total AMPKα in Huh7 cell transfected with siRNA against PRKAA1. (E,F) Relative mRNA expression of gluconeogenic genes (PEPCK, G6Pase; (E) and lipogenic genes (ACAT2, ACACA and FASN; (F) with or without knockdown of PRKAA1, followed by treatment with 50 nM pyrvinium for 24 h. * p < 0.05; ** p < 0.01. Data are representative of at least three independent experiments.

Figure 5

Effect of AMPK deletion on gluconeogenic and lipogenic gene expression in mouse embryonic fibroblasts (MEF) cells. (A) Protein expression of phosphorylated and total AMPK in AMPKα1/α2 wild-type (WT) and double knockout (DKO) MEF cells. (B–F) Relative mRNA expression of gluconeogenic genes G6Pase (B), Pepck (C) and lipogenic genes Acat2 (D), Acaca (E), Fasn (F) in AMPKα1/α2 WT and DKO MEF cells treated without and with 100nM pyrvinium for 24 h. Control: WT MEF cells without pyrvinium treatment. * p < 0.05; ** p < 0.01. Data are representative of at least three independent experiments.

Figure 6

Pyrvinium effect on β-catenin level was independent of its effect on AMPK phosphorylation. (A) β-catenin and AMPK phosphorylation in L-cells and L-Wnt3a cells cultured for 24 h. (B) Effect of β-catenin upregulation on pyrvinium-induced AMPK activation in HEK293 cells. The cells were cultured with and without WNT3a-conditioned medium and further treated with 100 nM pyrvinium for 24 h. (C) Protein expression of β-catenin in AMPKα1/α2 WT and DKO MEF cells treated with and without 100 nM pyrvinium for 24 h. Data are representative of at least three independent experiments.

Figure 7

In vivo efficacy of pyrvinium in treatment of metabolic disorders in mice. (A) Glucose tolerance test in high fat diet (HFD) and normal chow diet (NCD)-fed mice treated with and without pyrvinium. C57BL/6J mice (n = 5 per group) were treated with pyrvinium for 1 month and fasted for 6 h prior to intraperitoneal injection of 1g/kg glucose for glucose tolerance test. The dose of pyrvinium was gradually escalated from 0.2 mg/kg to 0.5 mg/kg in a span of one month. (B) Body weight monitoring of the mice. Injection arrow indicates the start of pyrvinium administration. (C) Haematoxylin and eosin staining of liver tissues (20X image, scale bar is 400μm). (D) Effect of pyrvinium on serum cholesterol levels. Serum was collected postmortem. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01.