Reactive oxygen species-induced TXNIP drives fructose-mediated hepatic inflammation and lipid accumulation through NLRP3 inflammasome activation

Abstract

Aims: Increased fructose consumption predisposes the liver to nonalcoholic fatty liver disease (NAFLD), but the mechanisms are elusive. Thioredoxin-interacting protein (TXNIP) links oxidative stress to NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation and this signaling axis may be involved in fructose-induced NAFLD. Here, we explore the role of reactive oxygen species (ROS)-induced TXNIP overexpression in fructose-mediated hepatic NLRP3 inflammasome activation, inflammation, and lipid accumulation.

Results: Rats were fed a 10% fructose diet for 8 weeks and treated with allopurinol and quercetin during the last 4 weeks. Five millimolars of fructose-exposed hepatocytes (primary rat hepatocytes, rat hepatic parenchymal cells [RHPCs], HLO2, HepG2) were co-incubated with antioxidants or caspase-1 inhibitor or subjected to TXNIP or NLRP3 siRNA interference. Fructose induced NLRP3 inflammasome activation and pro-inflammatory cytokine secretion, janus-activated kinase 2/signal transducers and activators of transcription 3-mediated inflammatory signaling, and expression alteration of lipid metabolism-related genes in cultured hepatocytes and rat livers. NLRP3 silencing and caspase-1 suppression blocked these effects in primary rat hepatocytes and RHPCs, confirming that inflammasome activation alters hepatocyte lipid metabolism. Hepatocellular ROS and TXNIP were increased in animal and cell models. TXNIP silencing blocked NLRP3 inflammasome activation, inflammation, and lipid metabolism perturbations but not ROS induction in fructose-exposed hepatocytes, whereas antioxidants addition abrogated TXNIP induction and diminished the detrimental effects in fructose-exposed hepatocytes and rat livers.

Innovation and conclusions: This study provides a novel mechanism for fructose-induced NAFLD pathogenesis by which the ROS-TXNIP pathway mediates hepatocellular NLRP3 inflammasome activation, inflammation and lipid accumulation. Antioxidant-based interventions can inhibit the ROS-TXNIP pathway.

FIG. 1.

Excess fructose intake induces NLRP3 inflammasome activation and IL-1β and IL-18 production in rat livers. Rats were given drinking water with or without 10% fructose for 8 weeks, after which liver biopsies were obtained for analysis. (A) Western blot analysis of NLRP3, ASC, and caspase-1 protein levels (n=8/group). (B) ELISA-based quantification of IL-1β and IL-18 levels (n=8/group). (C) Immunohistochemistry of NLRP3 (red) in liver sections co-stained with fluorescently labeled antibodies against CK18 (hepatocytes, green) or against glucocorticoid-inducible member of the scavenger receptor cysteine-rich family (CD163, Kupffer cells, green). Scale bar=100 μm. Results are expressed as mean±SEM. ^#p<0.05, ^###p<0.001 versus control group. ASC, apoptosis-associated speck-like protein; CK18, cytokeratin 18; IL, interleukin; NLRP3, NOD-like receptor family, pyrin domain containing 3. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

Fructose exposure stimulates NLRP3 inflammasome activation and IL-1β and IL-18 secretion in cultured hepatocytes. (A) Primary rat hepatocytes, RHPCs, HLO2, and HepG2 cells were cultured with or without 5 mM fructose for 72 h and analyzed for NLRP3, ASC, and caspase-1 protein levels by Western blot (n=8/group). (B) ELISA-based quantification of IL-1β and IL-18 levels in cell lysate (top) and supernatant (bottom) (n=8/group). (C) Primary rat hepatocytes and RHPCs were co-incubated with or without 5 mM fructose or/and the caspase-1-specific inhibitor Ac-YVAD-CMK for 72 h. Caspase-1 activity and IL-1β and IL-18 levels (ELISA) were determined in cell lysate (top) and supernatant (bottom) (n=8/group). (D) Primary rat hepatocytes and RHPCs were transfected with or without NLRP3-specific siRNA for 48 h, after which NLRP3 was assayed by RT-PCR (n=4/group). (E) Primary rat hepatocytes and RHPCs were transfected with NLRP3-specific siRNA for 48 h and co-incubated with 5 mM fructose for another 72 h. Western blot analysis of caspase-1 protein levels and ELISA-based determination of IL-1β and IL-18 levels in cell lysate (top) and supernatant (bottom) are shown (n=8/group). Results are expressed as mean±SEM. ^#p<0.05, ^##p<0.01, ^###p<0.001 versus control group. Ac-YVAD-CMK, Ac-Tyr-Val-Ala-Asp-chloromethylketone; RHPCs, rat hepatic parenchymal cells; RT-PCR, reverse transcription–polymerase chain reaction.

FIG. 3.

NLRP3 inflammasome activation alters lipid metabolism-related gene expression in fructose-exposed cultured hepatocytes. (A) Primary rat hepatocytes, RHPCs, HLO2, and HepG2 cells were cultured with or without 5 mM fructose for 72 h, after which p-JAK2, JAK2, p-STAT3, STAT3, and SOCS3 protein levels were assayed by Western blot (n=8/group). (B) Western blot analysis of PPAR-α, SREBP1, and SCD1 protein levels (n=8/group). (C) Primary rat hepatocytes and RHPCs were co-incubated with or without 5 mM fructose and caspase-1-specific inhibitor Ac-YVAD-CMK for 72 h, after which p-JAK2, JAK2, p-STAT3, STAT3, SOCS3, PPAR-α, SREBP1, and SCD1 protein levels were analyzed by Western blot. (D) Primary rat hepatocytes and RHPCs were transfected with NLRP3-specific siRNA for 48 h and co-incubated with or without 5 mM fructose for another 72 h, followed by Western blot analysis of p-JAK2, JAK2, p-STAT3, STAT3, SOCS3, PPAR-α, SREBP1, and SCD1 protein levels. Results are expressed as mean±SEM. ^#p<0.05, ^##p<0.01, ^###p<0.001 versus control group. JAK2, janus-activated kinase 2; PPAR-α, peroxisome proliferator-activated receptor-α; SCD1, stearoyl-CoA desaturase 1; SOCS3, suppressors of cytokine signaling 3; SREBP1, sterol regulatory element binding protein 1; STAT3, signal transducer and activator of transcription 3.

FIG. 4.

Fructose induces TXNIP overexpression in rat livers and cultured hepatocytes. Rats were given drinking water with or without 10% fructose for 8 weeks, after which liver biopsies were obtained for analysis. (A) Hepatic TXNIP protein levels were determined by Western blot (n=8/group). (B) Representative immunohistological sections stained for TXNIP (red) and co-stained for CK18 (hepatocytes, green) or CD163 (Kupffer cells, green). Scale bar=100 μm. (C) Primary rat hepatocytes, RHPCs, HLO2, and HepG2 cells were cultured with or without 5 mM fructose for 72 h, followed by Western blot analysis of TXNIP protein levels (n=8/group). Results are expressed as mean±SEM. ^##p<0.01, ^###p<0.001 versus control group. TXNIP, thioredoxin-interacting protein. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

TXNIP mediates fructose-induced NLRP3 inflammasome activation and lipid metabolism-related gene deregulation in cultured hepatocytes. (A) Primary rat hepatocytes, RHPCs, HLO2, and HepG2 cells were transfected with or without TXNIP siRNA for 48 h. TXNIP expression levels were analyzed by RT-PCR (n=4/group). (B) Primary rat hepatocytes, RHPCs, HLO2, and HepG2 cells were transfected with TXNIP siRNA for 48 h and co-incubated with or without 5 mM fructose for another 72 h. NLRP3, ASC, and caspase-1 protein levels were quantitated by Western blot (n=8/group). (C) IL-1β and IL-18 concentrations in cell lysate (top) and supernatant (bottom) were determined by ELISA (n=8/group). (D) Western blot analysis of PPAR-α, SREBP1, and SCD1 protein levels (n=8/group). (E) Primary rat hepatocytes and RHPCs were transfected with NLRP3 siRNA for 48 h and co-incubated with or without 5 mM fructose for another 72 h. TXNIP protein levels were measured by Western blot (n=8/group). (F) Primary rat hepatocytes and RHPCs were co-incubated with or without 5 mM fructose and the caspase-1 specific inhibitor Ac-YVAD-CMK for 72 h, after which TXNIP protein levels were analyzed by Western blot (n=8/group). Results are expressed as mean±SEM. ^#p<0.05, ^##p<0.01, ^###p<0.001 versus control group.

FIG. 6.

Fructose induces TXNIP overexpression through ROS production in hepatocytes. (A) Primary rat hepatocytes, RHPCs, HLO2, and HepG2 cells were cultured with or without 5 mM fructose for 48 h and co-incubated with 2 μM allopurinol or 20 μM quercetin for another 24 h. Total ROS, H₂O₂, O₂^•−, MDA, iNOS, XO, SOD, and TAC level or activity were analyzed (n=8/group). (B) TXNIP protein levels were determined in these hepatocytes by Western blot (n=8/group). (C) Primary rat hepatocytes and RHPCs were cultured with or without 5 mM fructose for 48 h and co-incubated with 100 μM tempol, 50 U/ml catalase, 100 μM vitamin E, 2 μM allopurinol, 20 μM quercetin, 100 μM apocynin, or 100 μM aminoguanidine for another 24 h. Total ROS, MMP, and NOX level or activity were analyzed (n=8/group). (D) Primary rat hepatocytes and RHPCs were transfected with TXNIP siRNA for 48 h, co-incubated with or without 5 mM fructose for 48 h, and treated with 2 μM allopurinol or 20 μM quercetin for another 24 h. Total ROS levels were measured (n=8/group). (E) Primary rat hepatocytes and RHPCs were transfected with NLRP3 siRNA for 48 h, co-incubated with or without 5 mM fructose for 48 h, and treated with 2 μM allopurinol or 20 μM quercetin for another 24 h. Total ROS levels were measured (n=8/group). (F) Primary rat hepatocytes and RHPCs were transfected with NLRP3 siRNA for 48 h, co-incubated with or without 5 mM fructose for another 48 h, and treated with 2 μM allopurinol or 20 μM quercetin for another 24 h. TXNIP protein levels were measured by Western blot (n=8/group). (G) Primary rat hepatocytes and RHPCs were co-incubated with or without 5 mM fructose and the caspase-1 specific inhibitor Ac-YVAD-CMK for 48 h and treated with 2 μM allopurinol or 20 μM quercetin for another 24 h. TXNIP protein levels were determined by Western blot (n=8/group). (H) Primary rat hepatocytes and RHPCs were cultured with or without 5 mM fructose for 48 h and treated with 2 μM allopurinol or 20 μM quercetin for another 24 h. Total and nuclear Nrf2 protein levels were assayed by Western blot (n=8/group). (I) Rats were given drinking water containing 10% fructose for 4 weeks and then given 5 mg/kg allopurinol or 50 or 100 mg/kg quercetin for another 4 weeks. Total and nuclear Nrf2 protein levels were determined in liver biopsies by Western blot (n=8/group). Results are expressed as mean±SEM. ^#p<0.05, ^##p<0.01, ^###p<0.001 versus control group; *p<0.05, **p<0.01, ***p<0.001 versus fructose vehicle group. iNOS, inducible nitric oxide synthase; MDA, malondialdehyde; MMP, mitochondrial membrane potential; Nrf2, nuclear factor (erythroid-derived-2)-like 2; NOX, NADPH oxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; TAC, total antioxidant capacity; XO, xanthine oxidase.

FIG. 6.

Fructose induces TXNIP overexpression through ROS production in hepatocytes. (A) Primary rat hepatocytes, RHPCs, HLO2, and HepG2 cells were cultured with or without 5 mM fructose for 48 h and co-incubated with 2 μM allopurinol or 20 μM quercetin for another 24 h. Total ROS, H₂O₂, O₂^•−, MDA, iNOS, XO, SOD, and TAC level or activity were analyzed (n=8/group). (B) TXNIP protein levels were determined in these hepatocytes by Western blot (n=8/group). (C) Primary rat hepatocytes and RHPCs were cultured with or without 5 mM fructose for 48 h and co-incubated with 100 μM tempol, 50 U/ml catalase, 100 μM vitamin E, 2 μM allopurinol, 20 μM quercetin, 100 μM apocynin, or 100 μM aminoguanidine for another 24 h. Total ROS, MMP, and NOX level or activity were analyzed (n=8/group). (D) Primary rat hepatocytes and RHPCs were transfected with TXNIP siRNA for 48 h, co-incubated with or without 5 mM fructose for 48 h, and treated with 2 μM allopurinol or 20 μM quercetin for another 24 h. Total ROS levels were measured (n=8/group). (E) Primary rat hepatocytes and RHPCs were transfected with NLRP3 siRNA for 48 h, co-incubated with or without 5 mM fructose for 48 h, and treated with 2 μM allopurinol or 20 μM quercetin for another 24 h. Total ROS levels were measured (n=8/group). (F) Primary rat hepatocytes and RHPCs were transfected with NLRP3 siRNA for 48 h, co-incubated with or without 5 mM fructose for another 48 h, and treated with 2 μM allopurinol or 20 μM quercetin for another 24 h. TXNIP protein levels were measured by Western blot (n=8/group). (G) Primary rat hepatocytes and RHPCs were co-incubated with or without 5 mM fructose and the caspase-1 specific inhibitor Ac-YVAD-CMK for 48 h and treated with 2 μM allopurinol or 20 μM quercetin for another 24 h. TXNIP protein levels were determined by Western blot (n=8/group). (H) Primary rat hepatocytes and RHPCs were cultured with or without 5 mM fructose for 48 h and treated with 2 μM allopurinol or 20 μM quercetin for another 24 h. Total and nuclear Nrf2 protein levels were assayed by Western blot (n=8/group). (I) Rats were given drinking water containing 10% fructose for 4 weeks and then given 5 mg/kg allopurinol or 50 or 100 mg/kg quercetin for another 4 weeks. Total and nuclear Nrf2 protein levels were determined in liver biopsies by Western blot (n=8/group). Results are expressed as mean±SEM. ^#p<0.05, ^##p<0.01, ^###p<0.001 versus control group; *p<0.05, **p<0.01, ***p<0.001 versus fructose vehicle group. iNOS, inducible nitric oxide synthase; MDA, malondialdehyde; MMP, mitochondrial membrane potential; Nrf2, nuclear factor (erythroid-derived-2)-like 2; NOX, NADPH oxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; TAC, total antioxidant capacity; XO, xanthine oxidase.

FIG. 7.

Antioxidants allopurinol and quercetin block NLRP3 inflammasome activation, IL-1β and IL-18 secretion, the JAK2/STAT3 pathway, and deregulated lipid metabolism in fructose-exposed cultured hepatocytes. Primary rat hepatocytes, RHPCs, HLO2, and HepG2 cells were exposed to 5 mM fructose or vehicle and co-incubated with 2 μM allopurinol or 20 μM quercetin for another 24 h. (A) NLRP3, ASC, and caspase-1 protein levels were analyzed by Western blot (n=8/group). (B) IL-1β and IL-18 levels were determined in cell lysate (top) and supernatant (bottom) (n=8/group). (C) p-JAK2, JAK2, p-STAT3, STAT3, and SOCS3 protein levels were assayed by Western blot (n=8/group). (D) PPAR-α, SREBP1, and SCD1 protein levels were determined by Western blot (n=8/group). Results are expressed as mean±SEM. ^#p<0.05, ^##p<0.01, ^###p<0.001 versus control group; *p<0.05, **p<0.01, ***p<0.001 versus fructose vehicle group.

FIG. 7.

Antioxidants allopurinol and quercetin block NLRP3 inflammasome activation, IL-1β and IL-18 secretion, the JAK2/STAT3 pathway, and deregulated lipid metabolism in fructose-exposed cultured hepatocytes. Primary rat hepatocytes, RHPCs, HLO2, and HepG2 cells were exposed to 5 mM fructose or vehicle and co-incubated with 2 μM allopurinol or 20 μM quercetin for another 24 h. (A) NLRP3, ASC, and caspase-1 protein levels were analyzed by Western blot (n=8/group). (B) IL-1β and IL-18 levels were determined in cell lysate (top) and supernatant (bottom) (n=8/group). (C) p-JAK2, JAK2, p-STAT3, STAT3, and SOCS3 protein levels were assayed by Western blot (n=8/group). (D) PPAR-α, SREBP1, and SCD1 protein levels were determined by Western blot (n=8/group). Results are expressed as mean±SEM. ^#p<0.05, ^##p<0.01, ^###p<0.001 versus control group; *p<0.05, **p<0.01, ***p<0.001 versus fructose vehicle group.

FIG. 8.

Allopurinol and quercetin reduce TXNIP overexpression and NLRP3 inflammasome activation and ameliorate inflammation and the extent of lipid metabolism deregulation in livers of fructose-fed rats. Rats were given drinking water containing 10% fructose for 4 weeks and subsequently 5 mg/kg allopurinol or 50 or 100 mg/kg quercetin for another 4 weeks. (A) Hepatic TXNIP protein levels were determined by Western blot (n=8/group). (B) Hepatic NLRP3, ASC, and caspase-1 protein levels were measured by Western blot (n=8/group). (C) Hepatic IL-1β and IL-18 levels were determined by ELISA (n=8/group). (D) Western blot analysis of hepatic p-JAK2, JAK2, p-STAT3, STAT3, and SOCS3 protein levels (n=8/group). (E) Hepatic PPAR-α, SREBP1, and SCD1 protein levels were determined by Western blot (n=8/group). Results are expressed as mean±SEM. ^#p<0.05, ^##p<0.01, ^###p<0.001 versus control group; *p<0.05, **p<0.01, ***p<0.001 versus fructose vehicle group.

FIG. 9.

Allopurinol and quercetin reduce TXNIP overexpression and NLRP3 inflammasome activation and ameliorate inflammation and the extent of lipid metabolism deregulation in livers of high-fat diet-fed rats. Rats were fed a high-fat diet for 4 weeks and subsequently given 5 mg/kg allopurinol or 50 or 100 mg/kg quercetin for another 4 weeks. (A) Representative images of liver sections immunohistochemically stained for NLRP3 (red) and CK18 (hepatocytes, green) or CD163 (Kupffer cells, green). Scale bar=100 μm. (B) Hepatic IL-1β and IL-18 levels were determined by ELISA (n=8/group). (C) Western blot analysis of NLRP3, ASC, caspase-1, TXNIP, and total and nuclear Nrf2 protein levels in rat livers (n=8/group). (D) Representative images of liver sections immunohistochemically stained for TXNIP (red) and CK18 (hepatocytes, green) or CD163 (Kupffer cells, green). Scale bar=100 μm. Results are expressed as mean±SEM. ^#p<0.05, ^##p<0.01 versus control group; *p<0.05, **p<0.01 versus high-fat diet vehicle group. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 10.

Induction of TXNIP overexpression through ROS production underlies fructose-induced hepatic inflammation and lipid accumulation. Fructose increased TXNIP expression through ROS production and nuclear Nrf2 suppression in hepatocytes, which, in turn, triggered NLRP3 inflammasome activation and secretion of IL-1β and IL-18. The consequent inflammatory signaling caused deregulation of lipid metabolism-related gene expression and lipid accumulation. These fructose-induced hepatic disturbances were blocked by the treatment of cells and livers with the antioxidants allopurinol and quercetin, which suppressed ROS levels.

FIG. 11.

Schematic diagram of the cell culture experiments and treatment regimens.