Overactivation of intestinal sterol response element-binding protein 2 promotes diet-induced nonalcoholic steatohepatitis

Abstract

Nonalcoholic fatty liver disease (NAFLD) is characterized by lipid accumulation in the liver that may progress to hepatic fibrosis and nonalcoholic steatohepatitis (NASH). Mechanisms underlying NAFLD and NASH are not yet fully understood. Dietary cholesterol was recently shown to be a risk factor for the development of NASH, suggesting a role for intestinal handling of cholesterol. One important regulator of cholesterol homeostasis is the sterol response element-binding protein-2 (SREBP-2) transcription factor. We tested the hypothesis that the overactivation of intestinal SREBP-2 increases the susceptibility to diet-induced NASH. A transgenic mouse model with intestine-specific overexpression of active SREBP-2 (ISR2 mice) driven by villin promoter was used. ISR2 mice and their wild-type littermates were fed a regular chow diet or a high-fat, high-cholesterol (HFHC) diet (15% fat, 1% cholesterol) for 15 wk. Results showed that HFHC feeding to ISR2 mice caused hepatic inflammation with increased levels of proinflammatory cytokines. Histological examination demonstrated extensive fibrosis after a HFHC diet associated with a perivascular as well as pericellular collagen deposits in ISR2 mice compared with wild-type littermates. The severe hepatic inflammation and advanced fibrosis in ISR2 mice was not associated with a difference in lipid accumulation in ISR2 mice compared with wild type littermates after HFHC feeding. These data indicate that overactivation of intestinal SREBP2 promotes diet-induced hepatic inflammation with features of human NASH resulting in rapid severe fibrosis and provide a novel link between regulatory processes of intestinal cholesterol and progression of fatty liver.NEW & NOTEWORTHY The current study highlights the role of overactivation of intestinal SREBP-2 transcription factor in the progression of hepatic fibrosis associated with diet-induced NASH. Mice with intestine-specific overexpression of SREBP-2 demonstrated more inflammation and severe fibrosis in the liver in response to 15 wk of being fed a high-cholesterol, high-fat diet as compared with their wild-type littermates. These data demonstrate a novel link between intestinal regulatory processes of cholesterol metabolism and the pathogenesis of fatty liver diseases.

Keywords: ISR2 mice; high cholesterol; high fat; intestinal sterol response element-binding protein 2; liver fibrosis; nonalcoholic fatty liver disease; nonalcoholic steatohepatitis.

Fig. 1.

Histological features of nonalcoholic steatohepatitis (NASH) in livers from ISR2 mice fed a high-fat, high-cholesterol (HFHC) diet. A: liver sections from ISR2 mice and their wild-type (WT) littermates stained with hematoxylin and eosin show inflammatory infiltrates and hepatocytes ballooning in ISR2 mice in response to 15 wk of HFHC feeding compared with regular chow diet (CD). B and C: severity of steatosis (B) and ballooning (C) was significantly increased in ISR2 mice and WT littermates after being fed HFHC diet compared with regular CD. D–F: total cholesterol (D), triglyceride (E), and the ratio of liver weight to body weight (F) were significantly increased in ISR2 and WT littermates after being fed a HFHC diet. G: the severity of inflammation as assessed from liver sections is significantly increased in ISR2 mice fed a HFHC diet compared with ISR2 fed a CD and to WT littermates fed with both diets. *Statistically significant difference compared with WT mice fed a regular CD; $statistically significant difference compared with WT mice fed a HFHC diet; #statistically significant difference compared with ISR2 mice fed a CD.

Fig. 2.

HFHC feeding increased the mRNA expression of inflammatory markers in ISR2 mice. Relative levels of mRNA of leukocyte common antigen CD45 (A), neutrophil marker Ly6G (B), macrophage marker F480 (C), and proinflammatory cytokines TNFα (D) and IL-1β (E), as well as the bacterial pathogen marker Toll-like receptor 2 (TLR-2; F), were significantly increased in ISR2 mice fed a HFHC diet compared with WT mice fed a regular CD and HFHC diet (n = 5–9 mice/group). NASH score of ISR2 mice fed with HFHC diet was significantly higher compared with their WT littermates fed HFHC or normal CD for 7- (G) and 15-wk (H) feeding. Plasma levels of alanine aminotransferase (ALT; I) and asparate aminotransferase (AST; J) were significantly increased in ISR2 mice in response to HFHC feeding compared with WT littermates. *Statistically significant difference compared with WT mice fed a regular CD; $statistically significant difference compared with WT mice fed a HFHC diet; #statistically significant difference compared with ISR2 mice fed a CD.

Fig. 3.

ISR2 mice fed a HFHC diet exhibit fibrosis and increased markers of liver damage. A: liver sections from ISR2 mice and their WT littermates after 15 wk of a HFHC diet stained with Sirius red. Top (X images): increased pericellular “chicken wire” fibrosis; bottom (Y images): a robust perivascular fibrosis in ISR2 mice. B: histological grading of fibrosis shows a significant increase in ISR2 mice fed a HFHC diet compared with WT littermates fed a regular CD. *Statistically significant difference compared with WT mice fed a regular CD; $statistically significant difference compared with WT mice fed a HFHC diet; #statistically significant difference compared with ISR2 mice fed a CD.

Fig. 4.

HFHC feeding increases the levels of collagen-1α (Col1α) and α-smooth muscle actin (α-SMA) in ISR2 mice. A: real-time RT-PCR showed a significant increase in the relative expression of Col1α mRNA levels in ISR2 mice fed a HFHC diet. B: the relative expression of α-SMA mRNA was significantly higher in ISR2 mice fed a CD compared with WT littermates and remained significantly higher after HFHC feeding. (n = 4–11 mice/group). C and D: Western blot analysis shows that the protein levels of Col1α (C) and α-SMA (D) were higher in ISR2 mice as compared with WT littermates after being fed a HFHC diet. E: relative expression of SMA protein/GAPDH in mice fed with HFHC diet. *Statistically significant difference compared with WT mice fed a regular CD; $statistically significant difference compared with WT mice fed a HFHC diet; #statistically significant difference compared with ISR2 mice fed a CD.

Fig. 5.

Expression of hepatic genes involved in bile acid and cholesterol metabolism. A: real-time RT-PCR demonstrated a significant decrease in the relative expression of HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) mRNA in ISR2 mice fed a CD compared with WT mice. This decrease persisted after a HFHC feeding and was also observed in WT mice after a HFHC feeding. B: the relative expression of LDLR mRNA was decreased in ISR2 mice compared with WT littermates fed a CD or HFHC diet. C: hepatic ABCG5 mRNA levels were significantly increased in ISR2 mice and in WT mice fed a HFHC diet compared with WT mice fed a CD. D: expression of CYP27A1 was significantly decreased in ISR2 mice fed a HFHC diet compared with WT mice fed a CD. E: data show that CYP7A1 mRNA was significantly decreased in ISR2 mice fed a HFHC diet compared with WT mice fed with CD and WT mice fed a HFHC diet. Data were collected from n = 5–10 mice/group. *Statistically significant difference compared with WT mice fed a regular CD; $statistically significant difference compared with WT mice fed a HFHC diet.

Fig. 6.

Schematic diagram for the link between intestinal sterol regulatory element-binding protein-2 (SREBP-2) overexpression and hepatic cholesterol as well as bile acid metabolism.