Endocrinol Metab.  2024 Feb;39(1):127-139. 10.3803/EnM.2023.1826.

FoxO6-Mediated TXNIP Induces Lipid Accumulation in the Liver through NLRP3 Inflammasome Activation

• Kim ME
• Lee JS
• Kim TW
• Park MH
• Kim DH

• KMID: 2552804
• DOI: http://doi.org/10.3803/EnM.2023.1826

Abstract

Background
Hepatic steatosis, which involves the excessive accumulation of lipid droplets in hepatocytes, presents a significant global health concern due to its association with obesity and metabolic disorders. Inflammation plays a crucial role in the progression of hepatic steatosis; however, the precise molecular mechanisms responsible for this process remain unknown.
Methods
This study investigated the involvement of the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome and the forkhead box O6 (FoxO6) transcription factor in the pathogenesis of hepatic steatosis. We monitored the NLRP3 inflammasome and lipogenesis in mice overexpressing the constitutively active (CA)-FoxO6 allele and FoxO6-null mice. In an in vitro study, we administered palmitate to liver cells overexpressing CA-FoxO6 and measured changes in lipid metabolism.
Results
We administered palmitate treatment to clarify the mechanisms through which FoxO6 activates cytokine interleukin (IL)-1β through the NLRP3 inflammasome. The initial experiments revealed that dephosphorylation led to palmitate-induced FoxO6 transcriptional activity. Further palmitate experiments showed increased expression of IL-1β and the hepatic NLRP3 inflammasome complex, including adaptor protein apoptotic speck protein containing a caspase recruitment domain (ASC) and pro-caspase-1. Furthermore, thioredoxin-interacting protein (TXNIP), a key regulator of cellular redox conditions upstream of the NLRP3 inflammasome, was induced by FoxO6 in the liver and HepG2 cells.
Conclusion
The findings of this study shed light on the molecular mechanisms underpinning the FoxO6-NLRP3 inflammasome axis in promoting inflammation and lipid accumulation in the liver.

Keyword

FoxO6; TXNIP; NLRP3 inflammasome; Interleukin-1beta; Hepatic steatosis

Figure

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  Fig. 1. Effects of forkhead box O6 (FoxO6) on cytokine production. Serum levels of pro-cytokines in mice injected with either constitutively active form of FoxO6 (FoxO6-CA) or null adenoviral vectors, assessed over a period of 13 days: enzyme-linked immunosorbent assay (ELISA) of (A) interleukin (IL)-1β, (B) IL-6, (C) tumor necrosis factor α (TNF-α), and (D) IL-10 (n=5 in each group). Three experiments were run for each protein, which yielded similar results; a single representative result is shown here for each protein. NS, not significant. aP<0.01 vs. the results obtained using the empty adenovirus vector by paired t test.

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  Fig. 2. Forkhead box O6 (FoxO6) regulates nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome and lipid accumulation in the liver of mice injected with an adenoviral vector expressing FoxO6. (A) Western blotting was used to detect FoxO6 in the nuclear extracts (20 μg of protein) of liver tissues. Bars in densitometry data represent mean±standard error, and significance was determined using the unpaired test non-trangenic (Ntg). (B) FoxO6 activated the NLRP3 inflammasome genes. Western blotting was used to detect NLRP3, apoptotic speck protein containing a caspase recruitment domain (ASC), caspase-1, interleukin (IL)-1β, and thioredoxin-interacting protein (TXNIP) in cytoplasmic extracts (20 μg of protein) from the liver. (C) Livers were stained with Oil Red O to visualize lipid accumulation (bar=100 μm). (D) Triglyceride levels in hepatic tissues. FoxO6-CA, constitutively active form of FoxO6. aP<0.01 vs. non-transgenic (n=3 each); bP<0.001 vs. empty vector virus by paired t test.

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  Fig. 3. Interaction between forkhead box O6 (FoxO6) and thioredoxin-interacting protein (TXNIP) induced interleukin (IL)-1β in palmitatetreated HepG2 cells. HepG2 cells were treated with palmitate at various concentrations, and FoxO6 levels were determined by Western blotting. Samples loaded on gels were probed with α-tubulin. (A) Levels of FoxO6 and IL-1β noticeably increased after treatment with 100 and 500 μM palmitate. (B) Effects of palmitate on IL-1β production. The conditioned medium from the experiment in (A) was used for the determination of IL-1β levels. (C) Western blotting showed that immunoprecipitated FoxO6 and TXNIP were physically associated with each other. (D) Levels of phosphoinositide 3-kinase (PI3K), phosphorylated protein kinase B (p-Akt), and total-Akt were noticeably diminished after treatment with 100 and 500 μM palmitate. (E) HepG2 cells were treated with palmitate (500 μM) for 24 hours. Cells were immunostained using rabbit anti-TXNIP antibody followed by immunoglobulin G conjugated with fluorescein isothiocyanate (green) (bar=50 μm). (F) β-Oxidation and lipogenesis genes were subjected to real-time quantitative reverse-transcription polymerase chain reaction analysis. IP, immunoprecipitation; IB, immunoblotting; PPARα, peroxisome proliferator-activated receptor alpha; ACO, acyl-coA oxidase; CPT, carnitine palmitoyltransferase; PPARγ, peroxisome proliferator-activated receptor gamma; ACC, acetyl-coA carboxylase; FASN, fatty acid synthase. aP<0.05 and bP< 0.01 vs. normal by paired t test; cP<0.001 vs. vehicle by one-way analysis of variance (ANOVA) with Tukey’s post hoc test.

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  Fig. 4. Palmitate-induced forkhead box O6 (FoxO6) regulated the nucleotide-binding oligomerization domain-like receptor pyrin domaincontaining- 3 (NLRP3) inflammasome in HepG2 cells. (A) Phosphorylated FoxO6 (p-FoxO6) and total-FoxO6 expression in nuclear. (B) Cytosolic levels of thioredoxin-interacting protein (TXNIP), caspase-1, and interleukin (IL)-1β noticeably increased in FoxO6-transfected (100 multiplicity of infection [MOI]) cells after treatment with 500 μM palmitate. (C) Triglyceride levels in liver cells. aP<0.01 vs. empty vector virus; bP<0.05 vs. FoxO6 virus by one-way analysis of variance (ANOVA) with Tukey’s post hoc test.

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  Fig. 5. Forkhead box O6 (FoxO6)-induced thioredoxin-interacting protein (TXNIP) regulated the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome. (A) Pre-treated (1 day) with or without TXNIP-small interfering RNA (siRNA; 20 nM), and then stimulated with FoxO6 (100 multiplicity of infection [MOI]) in cells, and analyzed by Western blotting. (B) FoxO6 bound to the TXNIP promoter in HepG2 cells transfected with a vector containing FoxO6 (100 MOI) or FoxO6-siRNA (100 MOI). After incubation for 24 hours, the cells were subjected to a chromatin immunoprecipitation (ChIP) assay using rabbit pre-immune immunoglobulin G (lanes 1, 2) or anti-FoxO6 antibody (lanes 3–6). The immunoprecipitates were subjected to polymerase chain reaction using the TXNIP promoter. (C) Effect of wild-type FoxO6 with or without TXNIP-siRNA (20 nM) on the activity of the interleukin (IL)-1β promoter. HepG2 cells in 48-well microplates were transduced with adenoviral vector (AdV)-FoxO6, TXNIP-siRNA (20 nM), or control AdV-null vectors at a fixed dose (MOI, 100 pfu/cell), followed by transfection with 0.5 μg of pcDNA and IL-1β DNA in the culture medium. After incubation for 24 hours, the cells were harvested and treated with palmitate (500 μM); luciferase and β-galactosidase activities were determined 8 hours after palmitate treatment. The relative luciferase activity was calculated based on the IL-1β-luciferase/β-galactosidase activity ratio. The data are expressed as mean±standard error of the mean. aP<0.001 vs. pcDNA treated cells; bP<0.01, cP<0.001 vs. IL-1β DNA treated cells by one-way analysis of variance (ANOVA) with Tukey’s post hoc test.

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  Fig. 6. Changes in the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome in response to forkhead box O6 (FoxO6) depletion. (A) Western blot was used to detect FoxO6, thioredoxin-interacting protein (TXNIP), NLRP3, apoptotic speck protein containing a caspase recruitment domain (ASC), caspase-1, and interleukin (IL)-1β in liver tissues; β-actin levels were used as loading controls. (B) The expression of relevant genes (FoxO6, insulin receptor substrate 1 [IRS1], IRS2, and IL-1β) was analyzed by quantitative polymerase chain reaction. The results were normalized with respect to actin levels. (C) Possible mechanism by which FoxO6 activates the NLRP3 inflammasome in insulin resistance. WT, wild-type; KO, knockout; TG, triglyceride; p, phospho; Ser, serine; Tyr, tyrosine; Akt, protein kinase B. aP<0.001 vs. WT by paired t test.

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