Canagliflozin ameliorates the development of NAFLD by preventing NLRP3-mediated pyroptosis through FGF21-ERK1/2 pathway

Abstract

Recent studies have suggested that sodium-glucose co-transporter2 inhibitors go beyond their glycemic advantages to ameliorate the development of NAFLD. However, little research has been done on the underlying mechanisms. Here, we took deep insight into the effect of canagliflozin (CANA), one of the sodium-glucose co-transporter2 inhibitor, on the progression of NAFLD, and explored the molecular mechanisms. Our findings showed that CANA-treated ob/ob and diabetic mice developed improved glucose and insulin tolerance, although their body weights were comparable or even increased compared with the controls. The CANA treatment ameliorated hepatic steatosis and lipid accumulation of free fatty acid-treated AML12 cells, accompanied by decreased lipogenic gene expression and increased fatty acid β oxidation-related gene expression. Furthermore, inflammation and fibrosis genes decreased in the livers of CANA-treated ob/ob and diabetic mice mice. FGF21 and its downstream ERK1/2/AMPK signaling decreased, whereas NLRP3-mediated pyroptosis increased in the livers of the ob/ob and diabetic mice mice, which was reversed by the CANA treatment. In addition, blocking FGF21 or ERK1/2 activity antagonized the effects of CANA on NLRP3-mediated pyroptosis in lipopolysaccharide plus nigericin-treated J774A.1 cells. We conclude that CANA treatment alleviated insulin resistance and the progression of NAFLD in ob/ob and diabetic mice mice independent of the body weight change. CANA protected against the progression of NAFLD by inhibiting NLRP3-mediated pyroptosis and enhancing FGF21-ERK1/2 pathway activity in the liver. These findings suggest the therapeutic potential of sodium-glucose co-transporter2 inhibitors in the treatment of NAFLD.

Figure 1

Study design. Body weight–matched ob/ob mice (n = 16) in 6-week-old were randomly assigned, into 2 groups (each with 8 mice). (1) Wild-type group: 8 male wild-type mice were fed on normal chow. (2) ob/ob+CANA group: CANA (10 mg/kg/d; oral gavage, n = 8) was given to ob/ob mice for 14 weeks and fed, on normal chow. (3) ob/ob group: 0.5% CMC-Na (same volume of CANA; oral gavage; n = 8) was given to, ob/ob mice for 14 weeks and fed on normal chow. Body weight–matched C57BL/6 mice (n = 25) were randomly divided into 2 groups. (1) NC group: 5 mice as normal controls fed on normal chow. (2) DM mice: another 20 more C57BL/6 mice were fed a HFD for 4 weeks, and then mice were given i.p. injections of 50 mg/kg streptozotocin over the course of 3 days to induce diabetes. Body weight–matched DM mice were randomly divided into 2 groups. (1) DM+CANA group: DM mice treated with CANA (10 mg/kg/d; oral gavage; n = 10) for 11 weeks and fed on HFD. (2) DM group: DM mice treated with, 0.5% CMC-Na (same volume of CANA; oral gavage; n = 10) for 11 weeks and fed on HFD. Abbreviations: CANA, canagliflozin; CMC-Na, sodium carboxymethyl cellulose solution; DM, diabetes mice; HFD, high-fat diet; NC, normal control; STZ, streptozotocin; WT, wild-type.

Figure 2

Effects of CANA on metabolism phenotype and insulin resistance. Both ob/ob and DM mice were treated as mentioned in the Materials and Methods section. Mice were kept in metabolic cages to record food intake (A, D), water intake (B, E), urine volume (C, F), and UALB/CREA ratio (G) during CANA treatment. (H and I) Body weight change in response to 16-week and 11-week CANA treatment in ob/ob and DM mice, respectively. (J and K) Fasting plasma glucose level. (L and M) Blood glucose levels and AUC during IPGTT in 8-week CANA treatment ob/ob mice and 10-week CANA treatment DM mice. (N) fasting serum insulin levels of, ob/ob mice assessed by ELISA. (O) Blood glucose levels and AUC during ITT in DM mice receiving 10-week CANA treatment. Data are presented as mean±SD (n = 4–8 in ob/ob mice, n = 5–10 in DM mice). *p <  0.05; **p <  0.01; ***p <  0.001. Abbreviations: CANA, canagliflozin; DM, diabetes mice; IGPTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test; NC, normal control; UALB/CREA, urine albumin to creatinine ratio; WT, wild-type.

Figure 3

Effects of CANA on lipid droplets accumulation and liver function in mice. Representative images of external liver appearance (A, C) and liver/body weight ratio (B, D) after CANA treatment in the ob/ob and DM mice, respectively. Representative images of Oil Red O staining (E, G) and hematoxylin and eosin staining (F, H) of liver sections. (I and J) Liver function evaluated by ALT, AST, and ALP levels. Data are presented as mean±SD (n = 4–8 in ob/ob mice, n = 5–10 in DM mice). *p < 0.05; **p < 0.01; ***p < 0.001. (K) Cell viability levels of AML12 cells after 0, 20, 40, 60, 80, and 100 μM CANA extract for 24 hours. Data are presented as mean±SD . *p < 0.05; **p < 0.01. (L) Graphical representation of, changes in Nile red staining in FFA-induced steatotic mouse AML12 cells. Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate amino-transferase; CANA, canagliflozin; DM, diabetic mice; FFA, free fatty acids; NC, normal control; WT, wild-type.

Figure 4

Related gene expressions of liver lipogenesis, fatty acid β-oxidation, inflammation, and fibrosis. Hepatic mRNA expression of lipogenesis (A, D), fatty acid β-oxidation (B, E), inflammation and fibrosis, genes in the ob/ob and DM mice (C, F). Representative images of Masson staining of liver tissue sections (G, H). Data are presented as mean±SD (n=4–8 in ob/ob mice, n = 5–10 in DM mice). *p <  0.05; **p <  0.01; ***p <  0.001. Abbreviations: ACC, acetyl-CoA carboxylase; ACOX1: acyl-coenzyme A oxidase 1; CANA, canagliflozin; CPT1α, carnitine palmitoyltransferase-1α; DM, diabetic mice; FASN, fatty acid synthase; MCP-1, monocyte chemoattractant protein-1; PGC1α, peroxisome proliferator-activated receptor γ coactivator 1α; PPARα, peroxisome proliferator-activated receptor α; SCD-1, stearoyl-CoA desaturase 1; SMA, smooth muscle actin; SREBP-1c, sterol regulatory element-binding protein-1c; WT, wild-type.

Figure 5

Effects of CANA on FGF21 signaling pathway in the liver and AML12 cells. (A and B) KEGG, pathway enrichment analysis of targets of different gene expression that were related to CANA-treated, ob/ob mice, and DM mice. (C and E) Expression of KLB, FGFR1, and FGF21 mRNA in the livers. (D and F) Serum, FGF21 levels were measured by ELISA in ob/ob (n = 4–8) and DM mice (n = 5–10). Representative western blot of ERK1/2, p-ERK1/2, AMPK, p-AMPK, mTOR and p-mTOR in the livers (G, H). FFA (0.75 mM) was, applied to AML12 cells for 24 hours in both the absence and presence of CANA (40 μM), PD166866 (10 μM) or, PD98059 (5 μM). (I–K) Representative western blot of ERK1/2, AMPK, and mTOR. Data are presented as mean±SD. *p <  0.05; **p <  0.01; ***p <  0.001. Abbreviations: CANA, canagliflozin; DM, diabetic mice; FFA, free fatty acids; FGF21, fibroblast growth factor 21; FGFR1, fibroblast growth factor 21 receptor 1; KLB, co-receptor β Klotho; NC, normal control; WT, wild-type.

Figure 6

Effects of CANA on NLRP3-related pyroptosis in the livers of ob/ob and DM mice. (A and B) Representative images of immunohistochemical staining for NLRP3 in mouse liver sections. Examples of NLRP3-positive hepatocytes (black arrows) and macrophages (red arrows). (C and D) Pyroptosis-related proteins, in liver tissues, including pro-caspase-1, cleaved-caspase-1, pro-GSDMD, cleaved GSDMD, and ASC, were, examined by western blot. (E and F) Immunofluorescence analysis of IL-1β (green). Abbreviations: CANA, canagliflozin; DM, diabetic mice; GSDMD, gasdermin D; NC, normal control; WT, wild-type.

Figure 7

Effects of CANA on NLRP3-related pyroptosis in J774A.1 cells treated with LPS and nigericin. (A) Cell viability levels of J774A.1 cells after 0, 5, 10, 20, 50, 100, and 200 μM CANA extract for 24 hours. Transmission electron microscopic images (B) and PI staining images. NLRP3, caspase-1, GSDMD (C), and ASC (D) protein expression levels of in J774A.1 cells. (E) Representative western blot of AMPK, p-AMPK, mTOR, and p-mTOR. (F) Immunofluorescence analysis of IL-1β (green) in J774A.1 cells. (G) Representative, western blot of NLRP3, caspase-1, GSDMD, and ASC in J774A.1 cells treated with LPS+nigericin, MCC950, and CANA. (H–J) NLRP3, caspase-1, GSDMD, and ASC in J774A cells from various treatment groups are, shown in this representative western blot. The cells were pretreated in the absence or presence of, PD166866 (10 μM), PD98059 (5 μM), and compound C (10 μM) for 1 hour, then treated for 6 hours with LPS, 1 hour for nigericin, and another 1 hour with CANA (10 μM) before LPS. Data are presented as mean±SD. *p < 0.05; **p < 0.01; ***p < 0.001. Abbreviations: CANA, canagliflozin; GSDMD, gasdermin D; LPS, lipopolysaccharide; PI, propidium iodide.