Caspase-1 as a central regulator of high fat diet-induced non-alcoholic steatohepatitis

Abstract

Nonalcoholic steatohepatitis (NASH) is associated with caspase activation. However, a role for pro-inflammatory caspases or inflammasomes has not been explored in diet-induced liver injury. Our aims were to examine the role of caspase-1 in high fat-induced NASH. C57BL/6 wild-type and caspase 1-knockout (Casp1(-/-)) mice were placed on a 12-week high fat diet. Wild-type mice on the high fat diet increased hepatic expression of pro-caspase-1 and IL-1β. Both wild-type and Casp1(-/-) mice on the high fat diet gained more weight than mice on a control diet. Hepatic steatosis and TG levels were increased in wild-type mice on high fat diet, but were attenuated in the absence of caspase-1. Plasma cholesterol and free fatty acids were elevated in wild-type, but not Casp1(-/-) mice, on high fat diet. ALT levels were elevated in both wild-type and Casp1(-/-) mice on high fat diet compared to control. Hepatic mRNA expression for genes associated with lipogenesis was lower in Casp1(-/-) mice on high fat diet compared to wild-type mice on high fat diet, while genes associated with fatty acid oxidation were not affected by diet or genotype. Hepatic Tnfα and Mcp-1 mRNA expression was increased in wild-type mice on high fat diet, but not in Casp1(-/-) mice on high fat diet. αSMA positive cells, Sirius red staining, and Col1α1 mRNA were increased in wild-type mice on high fat diet compared to control. Deficiency of caspase-1 prevented those increases. In summary, the absence of caspase-1 ameliorates the injurious effects of high fat diet-induced obesity on the liver. Specifically, mice deficient in caspase-1 are protected from high fat-induced hepatic steatosis, inflammation and early fibrogenesis. These data point to the inflammasome as an important therapeutic target for NASH.

Figure 1. Caspase-1 and IL-1β expression is increased after high fat feeding.

Livers of wild-type mice on the control or high fat diet were analyzed for pro-caspase-1 and IL-1β expression by immunoblot (A). Densitometric analysis was done for each protein normalized to HSC70 (B and C). mRNA for IL-1 expression in the liver of mice on the control or high fat diet was analyzed by RT-PCR (D). IL-1 was assessed by ELISA in whole liver tissue (E). Values with different superscripts are significantly different from one another (p<0.05). n = 4 control, n = 6 high fat.

Figure 2. Adiposity is increased in caspase-1 knockout mice.

Mice on the control and high fat diets were analyzed for body adiposity. Fat content of subcutaneous (B) and visceral (C) depots were calculated from three images. Total adipose volume was calculated from subcutaneous and visceral depots (D). Adiposity was normalized to body weight and expressed as% adipose (E). Lean mass was calculated based on the density of adipose tissue and normalized to body weight and expressed as% lean mass (F). Values represent means ± SEM. Values with different superscripts are significantly different from one another (p<0.05). n = 5 C57BL/6, n = 4 Casp1^-/-.

Figure 3. Caspase-1 knockout mice are protected from high fat-induced hepatic steatosis.

Representative images of H&E stained livers in wild-type and Casp1^-/- mice on control or high fat diet (10x) (A). NAFLD activity score of wild-type and Casp1^-/- mice on the high fat diet was assessed by histopathology of H&E stained livers in a blinded-fashion by BPG (B). Hepatic triglyceride (TG) levels were measured biochemically from mice on the control or high fat diets (C). Plasma alanine aminotransferase (ALT) levels were analyzed from mice on the control or high fat diets (D). Plasma cholesterol (E), FFA (F) and TG levels (G) were measured biochemically as described in the methods section. Values represent means ± SEM. Values with different superscripts are significantly different from one another (p<0.05). n = 4 control, n = 6 high fat.

Figure 4. High fat-induced expression of lipogenesis-related genes is attenuated in caspase-1 knockout mice.

Expression of mRNA for Pparγ, Srebp1c, Acc1, Fas and Scd1 were analyzed in the livers of wild-type and Casp1^-/- mice on high fat diets and normalized to 18S. Hepatic expression of phospho-ACC and total ACC were analyzed by western blot (A). Expression of Pparα, Aox, L-fabp, Cpt1α, Pgc1β, Lcad and Mcad were analyzed in the livers of wild-type and Casp1-/- mice on high fat diet and normalized to 18S (B). Apob and Mttp mRNA were measured (C) in the liver of wild-type compared to and Casp1-/- mice on control or high fat diets and normalized to 18S (C). Values represent means ± SEM. Values with + different superscripts are significantly different from wild-type mice on control diet one another (p<0.05). Values with * are significantly different from wild-type mice on high fat diet (p<0.05). n = 4 control, n = 6 high fat.

Figure 5. High fat-induced expression of inflammatory cytokines and chemokines is attenuated in caspase-1 knockout mice.

Inflammatory cytokine/chemokine production in the liver is associated the early stages of NASH. mRNA expression for Tnfα (A), Mcp-1 (B) and F4/80 (C) in the liver of wild-type mice on control or high fat diets was performed by RT-PCR. Values represent means ± SEM. Values with different superscripts are significantly different from one another (p<0.05). n = 4 control, n = 6 high fat.

Figure 6. High fat-induced early fibrogenesis is prevented in caspase-1 knockout mice.

Early to moderate fibrosis in the liver is associated with high fat diet feeding. αSMA immunohistochemistry (A, C), Sirius Red staining (B, D), Col1α1 mRNA (E) in the liver of mice on the control or high fat diets was performed by RT-PCR. Values represent means ± SEM. Values with different superscripts are significantly different from one another (p<0.05). n = 4 control, n = 6 high fat.