Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis

Abstract

Background & aims: Type 2 diabetes and nonalcoholic steatohepatitis (NASH) are associated with insulin resistance and disordered cholesterol homeostasis. We investigated the basis for hepatic cholesterol accumulation with insulin resistance and its relevance to the pathogenesis of NASH.

Methods: Alms1 mutant (foz/foz) and wild-type NOD.B10 mice were fed high-fat diets that contained varying percentages of cholesterol; hepatic lipid pools and pathways of cholesterol turnover were determined. Hepatocytes were exposed to insulin concentrations that circulate in diabetic foz/foz mice.

Results: Hepatic cholesterol accumulation was attributed to up-regulation of low-density lipoprotein receptor via activation of sterol regulatory element binding protein 2 (SREBP-2), reduced biotransformation to bile acids, and suppression of canalicular pathways for cholesterol and bile acid excretion in bile. Exposing primary hepatocytes to concentrations of insulin that circulate in diabetic Alms1 mice replicated the increases in SREBP-2 and low-density lipoprotein receptor and suppression of bile salt export pump. Removing cholesterol from diet prevented hepatic accumulation of free cholesterol and NASH; increasing dietary cholesterol levels exacerbated hepatic accumulation of free cholesterol, hepatocyte injury or apoptosis, macrophage recruitment, and liver fibrosis.

Conclusions: In obese, diabetic mice, hyperinsulinemia alters nuclear transcriptional regulators of cholesterol homeostasis, leading to hepatic accumulation of free cholesterol; the resulting cytotoxicity mediates transition of steatosis to NASH.

Figure 1. Increased hepatic cholesterol levels and LDLR expression in foz/foz mice with NASH

(A) Hepatic cholesteryl ester (CE) and (B) free cholesterol (FC) content in normal chow (0% [w/w] cholesterol) and HF (0.2% [w/w] cholesterol)-fed WT and foz/foz mice at 12- (□) and 24- (▪) weeks (n=5–10/grp – see METHODS) as determined by HPLC. (C) LDL receptor (LDLR) protein expression, normalized to heat-shock protein 90 (HSP90) expression. (D) Representative LDLR IHC staining from 24-week liver sections. Arrows indicate positive staining. Scale bars represent 50 μm. *P<0.05, vs. diet-matched control. ^#P<0.05, vs. genotype-matched control. ^†P<0.05, vs. time-matched control.

Figure 2. Decreased cholesterol and bile acid biosynthesis, and canalicular transporter gene expression in HF-fed foz/foz versus wildtype mice

(A) Microsomal HMG-CoA reductase activity at 12- (□) and 24-weeks (▪) in foz/foz and WT mice according to diet (values for n are in METHODS). (B) Hepatic acyl-CoA:cholesterol acyltransferase (ACAT)-2 protein, (C) cholesteryl ester hydrolase (CEH) mRNA, and (D) Cyp7a1 mRNA expression at 12-and 24-weeks. (E) Hepatic bile salt exporter protein (Bsep) and (F) ATP-binding cassette protein-G8 (ABCG8) protein expression at 12- and 24-weeks. Heat shock protein-90 (HSP90) (shown in panel F) was used as a loading control, but not shown in all panels for clarity (results for loading controls were similar). Same mice as in Figure 1. *P<0.05, vs. diet-matched control. ^#P<0.05, vs. genotype-matched control. ^†P<0.05, vs. time-matched control.

Figure 3. Effect of diet and genotype on hepatic expression of nuclear regulators involved in cholesterol homeostasis

(A) Sterol-response element binding protein-2 (SREBP-2), (B) liver-receptor homolog-1 (LRH-1), (C) farnesoid X-receptor (FXR), and (D) small heterodimer partner (Shp) expression at 12-(□) and 24- (▪) weeks was assessed using western blotting of isolated hepatic nuclear protein. TATA-box binding protein (TBP) (shown in panel B) was used as a loading control. Same mice as preceding figures. *P<0.05, vs. diet-matched control. ^#P<0.05, vs. genotype-matched control. ^†P<0.05, vs. time-matched control.

Figure 4. Insulin alters cholesterol-regulating protein expression in primary hepatocyte cultures

(A) Levels of sterol-response element binding protein-2 (SREBP-2), (B) low density lipoprotein receptor (LDLR), and (C) bile salt exporter protein (Bsep) expression in primary hepatocytes (whole cell lysates) treated with 0, 0.2, 6.5, and 13.0 ng/ml insulin for 48 h (n=3/grp). As shown in (D), protein expression was normalised to β-actin. (E) Liver receptor homolog-1 (LRH-1) and (F) small heterodimer partner (Shp) mRNA was assessed by RT-PCR. There was insufficient material to prepare nuclear protein extracts. This experiment was conducted three times with analyses in triplicate (n=9/grp total). *P<0.05 between groups.

Figure 5. Dietary cholesterol modulates hepatic cholesterol content and liver injury, but not other lipid profiles in NASH

(A) Serum alanine transaminase (ALT), (B) total hepatic cholesteryl ester (CE), and (C) hepatic free cholesterol (FC) content in WT (□) and foz/foz (▪) mice (n values as per METHODS) fed HF-diet containing 0, 0.2 or 2.0% (w/w) cholesterol for 24-weeks. (D) Hepatic TG, (E) diacylglycerides (DAG), and (F) total free fatty acids (FFA), as determined by HPLC. *P<0.05, vs. diet-matched control. ^#P<0.05, vs. genotype-matched, 0.0% cholesterol groups. ^†P<0.05, vs. genotype-matched, 0.2% cholesterol groups

Figure 6. Dietary cholesterol modulates hepatocyte apoptosis and macrophage recruitment in NASH

(A) Cell death, as assessed by cytokeratin-18 (Ck-18) fragmentation, and (B) macrophage cell recruitment (F4/80) were determined using IHC detection to (C) quantify positive cells (methods). (D) Quantification (ImageJ) of representative Sirius red-stained liver sections (E) from WT (□) and foz/foz (▪) mice (n values as per METHODS) fed HF-diet containing 0, 0.2 or 2.0% (w/w) cholesterol for 24-weeks. Arrows indicate positive staining. Same livers as Figure 5. Scale bars represent 20 μm (panel C) and 500 μm (panels E). *P<0.05, vs. diet-matched control. ^#P<0.05, vs. genotype-matched, 0.2% cholesterol groups. ^†P<0.05, vs. genotype-matched, 2.0% cholesterol groups