Knockout of sulfatase 2 is associated with decreased steatohepatitis and fibrosis in a mouse model of nonalcoholic fatty liver disease

Abstract

Sulfatase 2 (SULF2) is a heparan sulfate editing enzyme that regulates the milieu of growth factors and cytokines involved in a variety of cellular processes. We used a murine model of diet-induced steatohepatitis to assess the effect of SULF2 downregulation on the development of nonalcoholic steatohepatitis (NASH) and liver fibrosis. Wild-type B6;129 mice (WT) and Sulf2-knockout B6;129P2-SULF2^{Gt(PST111)Byg} mice (Sulf2-KO) were fed a fast-food diet (FFD) rich in saturated fats, cholesterol, and fructose or a standard chow diet (SC) ad libitum for 9 mo. WT mice on FFD showed a threefold increase in hepatic Sulf2 mRNA expression, and a 2.2-fold increase in hepatic SULF2 protein expression compared with WT mice on SC. Knockout of Sulf2 led to a significant decrease in diet-mediated weight gain and dyslipidemia compared with WT mice on FFD. Knockout of Sulf2 also abrogated diet-induced steatohepatitis and hepatic fibrosis compared with WT mice on FFD. Furthermore, expression levels of the profibrogenic receptors TGFβR2 and PDGFRβ were significantly decreased in Sulf2-KO mice compared with WT mice on FFD. Together, our data suggest that knockout of Sulf2 significantly downregulates dyslipidemia, steatohepatitis, and hepatic fibrosis in a diet-induced mouse model of NAFLD, suggesting that targeting of SULF2 signaling may be a potential therapeutic mechanism in NASH.NEW & NOTEWORTHY We report for the first time that in wild-type (WT) mice, fast-food diet (FFD) induced a threefold increase in hepatic Sulf2 mRNA and a 2.2-fold increase in sulfatase 2 (SULF2) protein expression compared with WT mice on standard chow diet (SC). We showed that knockout of SULF2 ameliorates FFD-induced obesity, hyperlipidemia, steatohepatitis, and fibrosis. These data, along with work from other laboratories, suggest that SULF2 may be critical to the ability of the liver to progress to nonalcoholic steatohepatitis and fibrosis in conditions of overnutrition.

Keywords: SULF2; fast-food diet; liver fibrosis; nonalcoholic fatty liver disease; nonalcoholic steatohepatitis; obesity; sulfatase.

Graphical abstract

Fig. 1.

Fast-food diet (FFD) increases sulfatase 2 (SULF2) expression. RT-PCR of hepatic Sulf2 mRNA expression normalized to 18s rRNA in wild-type (WT) mice fed standard chow diet (SC) or FFD for 9 mo (A). Western blot analysis of hepatic SULF2 protein expression normalized to GAPDH in WT mice fed SC or FFD (B). Total body weight (C) and liver-to-body weight ratio (D) of WT mice after 9 mo on ad libitum feeding with SC or FFD. Data shown represent 9–12 mice per group.

Fig. 2.

Weight gain and insulin homeostasis in mice. A: weight gain in wild-type (WT) mice and Sulf2-knockout (KO) mice on standard chow diet (SC) or fast-food diet (FFD) over the 9-mo study period. Data were further subcategorized by male and female sex and analyzed. Glucose-tolerance test (B) and insulin-tolerance test (C) in WT mice and Sulf2-KO mice on SC or FFD. Data shown represent 11 or 12 mice per group, or 5 or 6 mice per group when subcategorized by sex.

Fig. 3.

Fast-food diet (FFD)-induced dyslipidemia is ameliorated by sulfatase 2 (SULF2) knockout. A: frozen liver sections were stained with Oil Red O (ORO) stain. B: quantification of lipid staining using ImageJ software showed a trend toward decreased hepatic lipid deposition in Sulf2-knockout (KO) mice on FFD, compared with wild-type (WT) mice on the same diet. Triglyceride level (C), total cholesterol (D), and low-density lipoprotein (LDL) cholesterol (E) in WT and Sulf2-KO mice on SC or FFD. Data for total and LDL cholesterol are further subcategorized by male and female sex and analyzed. Data shown represent 9–12 mice per group or 4–6 mice per group when subcategorized by sex.

Fig. 4.

Short-hairpin RNA (shRNA)-mediated knockdown of sulfatase 2 (SULF2) ameliorates lipid accumulation in HepG2 cells. Lipid accumulation was assessed in HepG2 cells expressing shRNA targeting Sulf2 or scrambled shRNA, followed by incubation with 400 µM of palmitic acid or vehicle. BODIPY 493/503 lipid stain (A, green) and DAPI nucleic acid stain (B, blue) were assessed using confocal microscopy. Images of BODIPY and DAPI staining were merged (C). The number of lipid droplets per area (D) and average size of lipid droplets (E) in palmitic acid-treated HepG2 cells expressing scrambled vs. Sulf2 shRNA were assessed using ImageJ software.

Fig. 5.

Sulfatase 2 (SULF2) knockout (KO) decreases fast-food diet (FFD)-induced liver inflammation. A: serum alanine aminotransferase (ALT) levels in wild-type (WT) and Sulf2-KO mice on standard chow diet (SC) or FFD. RT-PCR of hepatic mRNA expression of TNFR1 (B), IL6R (C), IL1βR1 (D), and osteopontin (E) in WT and Sulf2-KO mice fed SC or FFD. The mRNA expression of each gene was normalized to 18s rRNA. Histological assessment of inflammation (F), ballooning (G), and steatosis (H) in WT vs. Sulf2-KO mice fed SC or FFD in hematoxylin and eosin-stained liver sections. Data shown represent 9–12 mice per group.

Fig. 6.

Sulfatase 2 (SULF2) decreases fast-food diet (FFD)-induced apoptosis and liver fibrosis. Sections of mouse liver tissue were stained to assess the degree of apoptosis and fibrosis in wild-type (WT) and Sulf2-KO mice on SC or FFD. A: TUNEL staining of liver tissue sections (4–7 mice per group on SC and 9–11 mice per group on FFD) to determine apoptosis. B: Masson’s trichrome-staining of liver tissue sections (11–12 mice per group) to assess liver fibrosis. C: Picrosirius red-stained liver tissue sections (11–12 mice per group) to determine collagen deposition. Quantification of stained area is shown underneath each group of slides.

Fig. 7.

Knockout (KO) of sulfatase 2 (SULF2) downregulates profibrogenic signaling. Hepatic mRNA levels of the profibrogenic genes TGFβR2 (A) and PDGFRβ (B) were assessed by quantitative RT-PCR in wild-type (WT) and Sulf2-KO mice on standard chow diet (SC) or fast food diet (FFD) and normalized to 18s rRNA in the same sample (8–12 mice/group). Protein levels of TGFβR2 (C) and PDGFRβ (D) were also assessed via Western blot analysis (7 mice per group). Representative Western blots depicting the protein of interest (top band) and GAPDH (bottom band) and their quantitation with ImageJ are shown.

Fig. 8.

TGFβR2- and PDGFRβ1-target genes are downregulated by sulfatase 2 (SULF2) knockout. Hepatic mRNA expression levels of Collagen 1α1 (A), TIMP1 (B), and α-SMA (C) were determined by quantitative RT-PCR in wild-type (WT) and Sulf2-KO mice on standard chow diet (SC) or fast-food diet (FFD) and normalized to 18s rRNA. Data were further subcategorized by sex and analyzed. Data shown represent 9–12 mice per group or 5 or 6 mice per group when subcategorized by sex.

Fig. 9.

Proposed model of positive feedback loop involving sulfatase 2 (SULF2), obesity, and metabolic syndrome in conditions of overnutrition. Fast-food diet predisposes mice to obesity and its metabolic complications (hyperlipidemia and insulin resistance) and creates conditions that favor upregulation of SULF2. Increased SULF2 further propagates obesity, hyperlipidemia, and insulin resistance in a positive feedback loop. Hepatic inflammation and fibrosis develop over time through a SULF2-dependent mechanism involving modulation of TGFβR2 and PDGFRβ1 signaling.