Regulation of hepatic fibrosis by carcinoembryonic antigen-related cell adhesion molecule 1

Abstract

Objective: NAFLD is a complex disease marked by cellular abnormalities leading to NASH. NAFLD patients manifest low hepatic levels of CEACAM1, a promoter of insulin clearance. Consistently, Cc1^-/- null mice displayed spontaneous hyperinsulinemia/insulin resistance and steatohepatitis. Liver-specific reconstitution of Ceacam1 reversed these metabolic anomalies in 8-month-old Cc1^-/-xliver+ mice fed a regular chow diet. The current study examined whether it would also reverse progressive hepatic fibrosis in mice fed a high-fat (HF) diet.

Methods: 3-Month-old mice were fed a high-fat diet for 3-5 months, and metabolic and histopathological analysis were conducted to evaluate their NASH phenotype.

Results: Reconstituting CEACAM1 to Cc1^-/- livers curbed diet-induced liver dysfunction and NASH, including macrovesicular steatosis, lobular inflammation, apoptosis, oxidative stress, and chicken-wire bridging fibrosis. Persistence of hepatic fibrosis in HF-fed Cc1^-/- treated with nicotinic acid demonstrated a limited role for lipolysis and adipokine release in hepatic fibrosis caused by Ceacam1 deletion.

Conclusions: Restored metabolic and histopathological phenotype of HF-fed Cc1^{-/-xliver+xliver+} assigned a critical role for hepatic CEACAM1 in preventing NAFLD/NASH including progressive hepatic fibrosis.

Keywords: Hyperinsulinemia; hepatic fibrosis; insulin clearance; insulin resistance; nonalcoholic fatty liver disease.

Fig. 1.

Expression and metabolic analysis. 3-month-old male mice were fed RD or HF diet for 3 months (n>5 per genotype/feeding group). (A) CEACAM1 protein level was analysed by immunoblotting (Ib) liver lysates with polyclonal antibodies against mouse (α-mCC1) or rat (α-rCC1) CEACAM1. To normalize against protein loading, the lower half of the membrane was immunoblotted with α-Tubulin. Numbers to the right of the gels indicate apparent molecular mass (kDa). Gels represent 2 experiments performed on 2 different mice/genotype. (B) mice were subjected to an intraperitoneal injection of insulin (0.75 U/kg BW) (panels i, iii), and glucose (1.5 g/kg BW) (panels ii and iv) to evaluate blood glucose levels at 0–180 or 0–120 min post-injection, respectively.

Fig. 2.

Histological and biochemical analysis. Livers of 6-month-old mice (n≥5/genotype) were extracted and sectioned for (A) H&E staining to detect inflammatory foci (yellow arrows) and microvesicular lipid droplets. Representative images are shown. (B) Hepatic fatty acid synthase (FASN) activity (panel i) and hepatic palmitate oxidation (panel ii) were assayed in overnight fasted RD-fed (upper graphs) and HF-fed mice (lower graphs). Assays were performed in triplicate. Values are expressed as means ± SEM. ^aP<0.05 vs Cc1^+/+; ^bP<0.05 vs L-CC1; and ^cP<0.05 vs Cc1^−/−. (C) Western blot analysis was performed on liver lysates by immunoblotting (Ib) with antibodies against phosphorylated signaling proteins (α-pNF-κB, α-pStat3, α-peNOS, α-pPKCζ) normalized against their non-phosphorylated counterparts immunodetected on parallel gels. Numbers to the right of the gels indicate apparent molecular mass (kDa). Gels represent 2 experiments performed on 2 different mice/genotype. (D) Tunnel staining on liver sections to detect apoptotic bodies(yellow arrows) in RD-fed and HF-fed mice. Representative images are shown. CHOP protein was detected by immunoblotting with α-CHOP antibody normalized against total protein (α-Tubulin) loaded on parallel gel. Numbers to the right of the gels indicate apparent molecular mass (kDa). Gels represent 2 experiments performed on different mice/genotype.

Fig. 3.

Analysis of of hepatic fibrosis. Mice were fed RD or HF for 3 months (n≥5/genotype/feeding group). (A) Sirius red staining on liver sections was performed to detect interstitial collagen deposition in the parenchyma of Cc1^−/− mice. Representative images are shown. Fibrosis scoring was evaluated by Brunt criteria in the accompanying table. (B) mRNA analysis of fibrotic markers in liver tissues from of RD or HF-fed mice normalized to Gapdh (RD) or 18S (HF). Values are expressed as means ± SEM. ^aP<0.05 vs Cc1^+/+; ^bP<0.05 vs L-CC1; and ^cP<0.05 vs Cc1^−/−. (C) Western blot analysis was performed on liver lysates by immunoblotting (Ib) with antibodies against phosphorylated signaling proteins (α-pSmad2, α-pSmad3) normalized against their non-phosphorylated counterparts immunodetected on parallel gels. Smad7 and SMA proteins were detected by immunoblotting with α-Smad7 or α-SMA antibodies normalized against Tubulin on parallel gels. Gels represent more than 2 experiments performed on different mice/genotype.

Fig. 4.

The effect of nicotinic acid on hepatic fibrosis. 3-month-old male mice were fed a HF diet for 5 months. (A) HF-fed mice were treated with vehicle (Veh) or once daily injection of nicotinic acid (NA) for 2 days before mice were sacrificed and blood and tissues were collected (n≥4 mice/genotype/treatment). Plasma (A) NEFA, (B) IL-6 and (C) insulin were measured in duplicate. Values are expressed as means ± SEM. ^aP<0.05 vs Cc1^+/+; ^bP<0.05 vs L-CC1; and ^cP<0.05 vs Cc1^−/−. (D) intraperitoneal Insulin tolerance test was used to assess insulin response, as in the legend to Fig. 1. *P<0.05 vs Cc1^−/−. (E) Livers of HF-fed NA-treated mice were sectioned for H&E staining to detect macrovesicular steatosis and inflammation (yellow arrows). Representative images are shown. (F) Sirius red staining of liver sections was used to detect collagen deposition in the parenchyma of mice. Representative images are shown. (G) mRNA analysis of representative genes involved in lipid metabolism, fibrosis, and inflammation in HF-fed NA-treated livers normalized to 18S. Values are expressed as means ± SEM. ^aP<0.05 vs Cc1^+/+; ^bP<0.05 vs L-CC1; and ^cP<0.05 vs Cc1^−/−. (H) Western blot analysis was performed on liver lysates as in the legend to Fig. 3.