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Wnt/β-catenin signaling activation promotes lipogenesis in the steatotic liver via physical mTOR interaction - PubMed

  • ️Sun Jan 01 2023

Wnt/β-catenin signaling activation promotes lipogenesis in the steatotic liver via physical mTOR interaction

Kewei Wang et al. Front Endocrinol (Lausanne). 2023.

Abstract

Background and aims: Wnt/β-catenin signaling plays an important role in regulating hepatic metabolism. This study is to explore the molecular mechanisms underlying the potential crosstalk between Wnt/β-catenin and mTOR signaling in hepatic steatosis.

Methods: Transgenic mice (overexpress Wnt1 in hepatocytes, Wnt+) mice and wild-type littermates were given high fat diet (HFD) for 12 weeks to induce hepatic steatosis. Mouse hepatocytes cells (AML12) and those transfected to cause constitutive β-catenin stabilization (S33Y) were treated with oleic acid for lipid accumulation.

Results: Wnt+ mice developed more hepatic steatosis in response to HFD. Immunoblot shows a significant increase in the expression of fatty acid synthesis-related genes (SREBP-1 and its downstream targets ACC, AceCS1, and FASN) and a decrease in fatty acid oxidation gene (MCAD) in Wnt+ mice livers under HFD. Wnt+ mice also revealed increased Akt signaling and its downstream target gene mTOR in response to HFD. In vitro, increased lipid accumulation was detected in S33Y cells in response to oleic acid compared to AML12 cells reinforcing the in vivo findings. mTOR inhibition by rapamycin led to a down-regulation of fatty acid synthesis in S33Y cells. In addition, β-catenin has a physical interaction with mTOR as verified by co-immunoprecipitation in hepatocytes.

Conclusions: Taken together, our results demonstrate that β-catenin stabilization through Wnt signaling serves a central role in lipid metabolism in the steatotic liver through up-regulation of fatty acid synthesis via Akt/mTOR signaling. These findings suggest hepatic Wnt signaling may represent a therapeutic strategy in hepatic steatosis.

Keywords: Wnt signaling; beta-catenin (B-catenin); fatty acid synthesis; hepatic steatosis; high fat diet.

Copyright © 2023 Wang, Zhang, Lehwald, Tao, Liu, Liu, Koh and Sylvester.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1

Wnt+ mice are more susceptible to liver steatosis under high fat diet. Wild-type and Wnt1 overexpressing (Wnt+) mice were fed with HFD or normal control diet for 12 weeks. (A) Immunoblot shows a significant increase in β-catenin and Cyclin D1 protein in Wnt+ livers. α-tubulin served as a loading control. Signals were semi-quantified with Image J software. (B) By abdominal inspection, Wnt+ mice demonstrated severe abdominal obesity. (C) Representative liver histology of untreated or HFD-treated livers. Severe hepatic steatosis is detected in Wnt+ mice after HFD. H&E staining reveals increased liver steatosis in β-catenin-stabilized livers with lipid accumulation and hepatocytes ballooning. Lipid droplets are detected by ORO staining. (D) The liver/body weight ratio was measured after HFD in WT and Wnt+ mice. A significantly increased liver/body weight ratio was observed in Wnt+ mice after HFD compared to WT controls. Data are expressed as means ± SD of three separate experiments. Ctrl control group, HFD high fat diet, WT Wild-type, Wnt+ Wnt1 overexpressing, H&E hematoxylin-eosin, ORO s oil red O staining. *p value<0.05, **p value<0.01.

Figure 2
Figure 2

Increased fatty acid synthesis in Wnt+ mice under high fat diet. (A) Immunoblot shows a significant increase in the expression of fatty acid synthesis-related genes (SREBP-1 and its downstream targets ACC, AceCS1, and FASN) and a decrease in fatty acid oxidation gene (MCAD) in Wnt+ livers under HFD. mTOR and its downstream gene Phospho-S6K were significantly increased in Wnt+ mice in response to HFD. α-tubulin served as a loading control. Increased serum cholesterol (B), triglycerides (C), and glucose (D) levels were found in HFD-treated Wnt+ mice when compared to wildtype. WT Wild-type, HFD high fat diet. *p value<0.05, **p value<0.01.

Figure 3
Figure 3

Activated Wnt/β-catenin signaling promotes steatosis in vitro under oleic acid. AML12 hepatocytes and β-catenin stabilized AML12 (S33Y) cells were treated with 150 nM OA for 24 hours for steatosis induction. (A) Increased lipid accumulation was detected in S33Y cells in response to OA treatment compared to AML12 cells as detected by ORO staining. (B) For quantification of intracellular lipids, ORO absorbance was measured by spectrophotometer. A significant increase of lipid accumulation in the OA-treated S33Y cells was observed compared to AML12 cells. (C) Increased fatty acid synthesis-related genes (PPRA γ, FASN) were up-regulated in S33Y hepatocytes after OA incubation for 24 hours. α-tubulin served as a loading control. (D) Cells were treated with the mTOR inhibitor rapamycin at 20 nM for one hour. Inhibition of mTOR led to decreased expression of p-S6K, PPAR-γ, and FASN as detected by immunoblot analysis. OA oleic acid. *p value<0.05, **p value<0.01, n.s., not significant.

Figure 4
Figure 4

β-catenin directly interacts with mTOR signaling to induce lipogenesis. (A) Increased expression of lipid synthesis-related genes (mTOR, p-S6K, FASN, and PPAR-γ) were detected by immunoblotting in response to OA when Wnt/β-catenin signaling was activated via Wnt3a or LiCl application (lane 4 vs. 3 and 6 vs. 5). (B) After adding IWR (IWR-1-endo, an inhibitor of Wnt-signaling) to AML12 cells, the expression of β-catenin and mTOR was down-regulated (lane 2 vs. 1 and 4 vs. 3). In addition, after Wnt signaling was inhibited, even under the effect of OA, the expression level of mTOR did not increase (lane 4 vs. 2). (C) β-catenin has a physical interaction with mTOR, which was verified by co-immunoprecipitation in hepatocytes using anti-mTOR antibody followed by β-catenin immunoblotting. LiCl lithium chloride. Data were shown as mean ± SD of three separate experiments. *P < 0.05, **P < 0.01, based on a Student’s t-test.

Figure 5
Figure 5

Proposed working model describing the possible role of Wnt/β-catenin signaling for hepatosteatosis. In the absence of Wnt-signal (Off-state), ubiquitination and proteasome degradation of β-catenin will occur. Under this condition, the effect of HFD on hepatic fat synthesis is minimal. In the presence of Wnt ligand (On-state), HFD can induce and promote the formation of β-catenin/mTOR complex in the liver, leading to the upregulation of lipid synthesis-related genes (p-S6K, FASN, and PPAR-γ), increasing the lipid synthesis, and the developing of hepatic steatosis finally. Ub ubiquitination, HFD high fat diet, P phosphorylation.

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