Niemann-Pick type C2 protein supplementation in experimental non-alcoholic fatty liver disease

Background and aims Hepatic cholesterol deposition drives inflammation and fibrosis in non-alcoholic steatohepatitis (NASH). The Niemann-Pick type C2 (NPC2) protein plays an important role in regulating intracellular cholesterol trafficking and homeostasis. We hypothesized that intravenous NPC2 supplementation reduces cholesterol accumulation, hepatic inflammation and fibrogenesis in a nutritional NASH rat model. Methods: Rats were fed a high-fat, high-cholesterol (HFHC) diet for four weeks resulting in moderately severe NASH. Animals were treated with intravenous NPC2 or placebo twice weekly for either the last two weeks or the entire four weeks. End-points were liver/body- and spleen/body weight ratios, histopathological NASH scores, fibrosis, serum liver enzymes, cholesterol, lipoproteins, cytokines, and quantitative polymerase chain reaction derived hepatic gene expression related to cholesterol metabolism, inflammation, and fibrosis. Results: HFHC rats developed hepatomegaly, non-fibrotic NASH histopathology, elevated liver enzymes, serum cholesterol, and pro-inflammatory cytokines. Their sterol regulatory element binding factor 2 (SREBF2) and low-density lipoprotein receptor (LDL-R) mRNAs were down-regulated compared with rats on standard chow. NPC2 did not improve liver weight, histopathology, levels of serum liver enzymes or pro-inflammatory tumor necrosis factor-α (TNFα), Interleukin (IL)-6, or IL-1β in HFHC rats. Two weeks of NPC2 treatment lowered hepatic TNFα and COL1A1 mRNA expression. However, this effect was ultimately reversed following additional two weeks of treatment. Four weeks NPC2 treatment of rats raised ATP-binding cassette A1 (ABCA1) and low-density lipoprotein receptor (LDLR) mRNAs in the liver, concurrent with a strong tendency towards higher serum high-density lipoprotein (HDL). Furthermore, the peroxisome proliferator activated receptor-ɣ (PPARG) gene expression was reduced. Conclusions: NPC2 proved inefficient at modifying robust hepatic NASH end-points in a HFHC NASH model. Nonetheless, our data suggest that hepatic ABCA1 expression and reverse cholesterol transport were upregulated by NPC2 treatment, thus presenting putative therapeutic effects in diseases associated with deregulated lipid metabolism

Hepatic gene expression related to inflammation, fibrogenesis and cholesterol metabolism.

<p>Relative mRNA expressions compared with isocaloric diet fed controls of <i>TNF</i> and <i>COL1A1</i> (Panel A), <i>TGFB1</i> and <i>PPARG</i> (Panel B), <i>SREBF2</i>, <i>LDLR</i>, <i>HMGCR</i> and <i>ABCA1</i> (Panel C) and <i>INSIG1</i>, <i>SRB1</i>, <i>GNMT</i> and <i>NPC2</i> (Panel D) in isocaloric controls, high-fat-high-cholesterol (HFHC) controls, two-week NPC2 (treatment) and four-week NPC2 (prevention) animals. Bars represent median ± interquartile range. *: P < 0.05 compared with Isocaloric Controls. **: P < 0.005 compared with Isocaloric controls. ***: P < 0.0005 compared with Isocaloric controls. #: P< 0.05 compared with HFHC Controls. ¤: P<0.05 compared with two-week NPC2.</p

Hepatic histology scores.

<p>Hepatic histology scores graded semi-quantitatively according to Kleiner et. al. non-alcoholic fatty liver disease (NAFLD) activity score (NAS), steatosis (0–3), lobular inflammation (0–2), ballooning (0–2) and fibrosis (0–4) in isocaloric controls, high-fat, high-cholesterol (HFHC) controls, two-week NPC2 (treatment) and four-week NPC2 (prevention) animals. No animal had significant histological fibrosis on HE (Panel A) or Masson trichrome staining (Panel B). *: P < 0.05 compared with Isocaloric Controls. #: P < 0.05 compared with HFHC Controls.</p

Relative gene expressions in NAFLD and NASH patients and in fibrotic and non-fibrotic NAFLD (F1/2 versus F0).

<p>Relative gene expressions in NAFLD and NASH patients and in fibrotic and non-fibrotic NAFLD (F1/2 versus F0).</p