Heparanase Inhibition Prevents Liver Steatosis in E0 Mice

Abstract

Background: Non-alcoholic fatty liver disease affects up to 30% of adults in the USA, and is associated with a higher incidence of chronic liver morbidity and mortality. Several molecular pathways are involved in the pathology of liver steatosis, including lipid uptake, lipogenesis, lipolysis, and beta-oxidation. The enzyme heparanase has been implicated in liver steatosis. Herein, we investigated the effect of heparanase inhibition on liver steatosis in E₀ mice.

Methods: In vivo experiments: Male wild-type mice fed with either chow diet (n = 4) or high-fat diet (n = 6), and male E₀ mice fed with chow diet (n = 8) or high-fat diet (n = 33) were included. Mice on a high-fat diet were treated for 12 weeks with PG545 at low dose (6.4 mg/kg/week, ip, n = 6) or high dose (13.3 mg/kg/week, ip, n = 7), SST0001 (1.2 mg/mouse/day, ip, n = 6), or normal saline (control, n = 14). Animals were sacrificed two days after inducing peritonitis. Serum was analyzed for biochemical parameters. Mouse peritoneal macrophages (MPMs) were harvested and analyzed for lipid content. Livers were harvested for histopathological analysis of steatosis, lipid content, and the expression of steatosis-related factors at the mRNA level. In vitro experiments: MPMs were isolated from untreated E₀ mice aged 8-10 weeks and were cultured and treated with either PG545 or SST0001, both at 50 µg/mL for 24 h, followed by assessment of mRNA expression of steatosis related factors.

Results: Heparanase inhibition significantly attenuated the development of liver steatosis, as was evident by liver histology and lipid content. Serum analysis indicated lowering of cholesterol and triglycerides levels in mice treated with heparanase inhibitors. In liver tissue, assessment of mRNA expression of key factors in lipid uptake, lipolysis, lipogenesis, and beta-oxidation exhibited significant downregulation following PG545 treatment and to a lesser extent when SST0001 was applied. However, in vitro treatment of MPMs with PG545, but not SST0001, resulted in increased lipid content in these cells, which is opposed to their effect on MPMs of treated mice. This may indicate distinct regulatory pathways in the system or isolated macrophages following heparanase inhibition.

Conclusion: Heparanase inhibition significantly attenuates the development of liver steatosis by decreasing tissue lipid content and by affecting the mRNA expression of key lipid metabolism regulators.

Keywords: E0 mice; PG545; SST0001; heparanase; liver steatosis.

Figure 1

Study methodology. (A) In vivo experiments. C57 ApoE^−/− mice were placed on CD or HFD; the latter were treated with PG545 in two doses, SST0001 or NS. C57 WT mice were placed on either CD or HFD. Mice were then sacrificed, and blood, livers, and MPMs were collected for analysis. (B) In vitro experiments. MPMs were collected from C57 ApoE^−/− mice, treated with PG545 or SST0001 and tested for lipid content and beta-oxidation of lipids.

Figure 2

Effect of heparanase inhibition on liver steatosis. H&E, Oil-Red-O, and Masson’s Trichrome stainings of liver sections from E₀ mice on CD (A–C), E₀ mice on HFD (D–F), E₀ mice on HFD and PG545 low dose (G–I), E₀ mice on HFD and PG545 high dose (J–L), and E₀ mice on HFD and SST0001 (M–O).

Figure 3

Effect of SST0001 on liver histology. Immunoflourescent staining of liver sections with macrophages marker (F4/80 antibody) and wheat germ agglutinin (WGA, a carbohydrate-binding lectin) showing granuloma-like structures.

Figure 4

Effect of heparanase inhibitors on lipid uptake in macrophages. Cholesterol, LDL-C, HDL-C, and TG serum levels (A–D), TG and cholesterol content in the liver (E,F), TG and cholesterol in MPMs in vivo (G,H), and liver enzymes in serum (I,J). * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 5

Effect of heparanase inhibitors on lipid metabolism in the liver. mRNA expression of lipid metabolism pathway markers in liver (A–H). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Effect of heparanase inhibitors on macrophage lipid uptake and inflammation (in vitro). TC and TG content (A,B), mRNA expression of inflammation markers (C–E) and lipid uptake and metabolism markers (F–N). (*) as compared to Control. (#) as compared to PG545. * p < 0.05, ** p < 0.01, *** p < 0.001. # p < 0.05, ## p < 0.01.

Figure 7

Effect of heparanase inhibition on lipid oxidation (A–D), inflammatory stress (E–G) and autophagy (H,I) in the liver. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 8

Effect of heparanase inhibtion on liver fibrosis. * p < 0.05, ** p < 0.01, *** p < 0.001.