TM4SF5-mediated abnormal food-intake behavior and apelin expression facilitate non-alcoholic fatty liver disease features

Figures

• 

  Figure 1. Hepatocyte-specific TGTm4sf5-Flag mice show abnormal food-intake behavior and increased apelin levels Nine-week-old male C57BL/6 mice (WT or TG Alb-TGTm4sf5−Flag, n = 4) were fed normal chow ad libitum for an entire day (NCD1w) or during the daytime (9:00 a.m. to 9:00 p.m., NCD-DF1w) for 7 days before analyses. (A) Body weight changes (BWG, Δg). (B) Daytime or nighttime food intakes (g/mouse/day) for each day. (C) Changes in daily food intake (g/mouse/cage) during the diet. (D) Plasma AST or ALT levels. (E) H&E staining of liver tissues. Scale of 60 μm. (F) Plasma levels of triglycerides, total cholesterol, and glucose. (G) Plasma levels of food intake regulators. (H) Tm4sf5, Apelin, and Spp-1/Opn mRNA levels in liver tissues. See Table 1 for the primers. (I) Immunoblots of liver tissue extracts for the indicated molecules. (J) mRNA analyses from mRNA-Seq data prepared from SNU449-EV (empty vector) and SNU449-TM4SF5 cells. ∗, ∗∗, and ∗∗∗ indicate p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively. Data are represented as mean ± SD. Data represent three independent experiments. See also Figure S1.

• 

  Figure 2. TM4SF5-dependent apelin expression is linked to the regulation of SPP-1/OPN expression via AKT1/ELK1 activity (A–C) SNU449 hepatocytes transfected with empty vector (EV), TM4SF5 (A, B), or HepG2 (A, C) or Huh7 (C) cells transfected with shRNA against non-specific (NS) or the TM4SF5 sequence (#2 or #4, see Table 2) were harvested, prior to immunoblots (A) or qRT-PCR (B, C) for the indicated molecules. ∗, ∗∗, and ∗∗∗ indicate p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively. Data are represented as mean ± SD. (D) Heatmap for molecules related to the apelin signaling pathway from the mRNA-Seq analysis using liver tissues of 3-month-old male WT, Alb-TGTm4sf5, and Tm4sf5−/− KO mice fed normal chow ad libitum. (E) SNU449 cells as in (A) were treated with apelin at different concentrations for 24 h before whole cell lysate preparation and immunoblots for the indicated molecules. (F) Cyclosporin A were treated to SNU449 cells (0 or 250 nM for 24 h), before whole cell harvests and immunoblots. (G) SNU449 cells stably expressing empty vector (EV) or TM4SF5 expression vector were transiently transfected with siRNA against control sequence (−) or ELK1 sequences (#2 or #3, see Table 2), before…

• 

  Figure 3. Alb-TGTm4sf5-Flag mice fed HFD showed greater food intake than KO mice Male WT, Alb-TGTm4sf5−Falg, and Tm4sf5−/− KO mice (n = 4) were fed high-fat diet (60% kcal fat, HFD1w) ad libitum during an entire day (i.e., 24 h) or daytime only (i.e., 9:00 a.m. to 9:00 p.m., HFD-DF1w) for 1 week. (A) Scheme for the experimental protocol. (B) Normalized body weights (g). (C) Food intake (g/mouse/day) for either daytime only, nighttime only, or an entire day (24 h) of animals fed HFD1w. (D) Daily food intake (g) of animals fed HFD1w or HFD-DF1w. (E) Plasma AST or ALT levels and liver/body weight ratio of the animals. (F) Plasma triglycerides, cholesterol, and glucose levels of the animals. (G and H) Levels of food intake regulators including apelin and SPP-1/OPN measured in animal plasma samples. ∗, ∗∗, and ∗∗∗ indicate p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively. ns indicates non-significance. Data are represented as mean ± SD. (I) Whole tissue extracts of livers from the animals fed HFD1w or HFD-DF1w were processed for immunoblots for the indicated molecules. Two pieces of a liver from an animal were processed for the analyses. The data shown represent three isolated experiments.

• 

  Figure 4. Alb-TGTm4sf5−Flag mice fed HFD ad libitum showed inflammatory livers (A and B) Male WT, Alb-TGTm4sf5−Falg, and Tm4sf5−/− KO mice (n = 4) were fed HFD1wad libitum during an entire day (i.e., 24 h) or daytime only (i.e., 9:00 a.m. to 9:00 p.m., HFD-DF1w) for 1 week before various analyses. Liver tissues were processed for qRT-PCR to measure the relative mRNA levels for the indicated molecules (A), immunoblots (B), or H&E staining and immunohistochemistry using anti-F4/80 antibody. (C) Scale of 100 μm. (D) Subconfluent TM4SF5-negative SNU449-EV or HepG2-shTM4SF5#4 and TM4SF5-positive SNU449-TM4SF5 or HepG2-shNS were harvested for immunoblots for the indicated molecules. ∗ and ∗∗ depict p ≤ 0.05 and p ≤ 0.01, respectively. Data are represented as mean ± SD. Yellow arrows indicate infiltrated immune cells, and red arrowheads indicate damaged hepatocytes. Data shown represent three isolated experiments.

• 

  Figure 5. Alb-TGTm4sf5−Falg mice fed HFD1w showed peroxisomal β-oxidation and mTOR activation (A–D) Male WT, Alb-TGTm4sf5−Flag, and Tm4sf5−/− KO mice (n = 4) were fed NCD1w or HFD1wad libitum for an entire day (i.e., 24 h) or daytime only (i.e., 9:00 a.m. to 9:00 p.m.), NCD-DF1w, or HFD-DF1w before analyses. Liver tissues of the animals were processed for qRT-PCR (A, C) or immunoblots (B, D) for the indicated FAO-related molecules. ∗, ∗∗, and ∗∗∗ depict p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively. ns indicates non-significance. Data are represented as mean ± SD. Data shown represent three independent experiments.

• 

  Figure 6. Apelin treatment of Alb-TGTm4sf5−Flag mice fed HFD2w promoted NAFLD features Male WT, Alb-TGTm4sf5−Falg, and Tm4sf5−/− KO mice (n = 4) were fed HFD2wad libitum during an entire day (i.e., 24 h) or daytime only (i.e., 9:00 a.m. to 9:00 p.m., HFD-DF2w) for 2 weeks with saline or apelin treatment (300 μg/injection/kg/day) via intraperitoneal injection before analyses. (A) Scheme of the experimental protocol. (B) Changes in body weights (%, body weight gains; BWG) of the animals. (C) Food intake (g/mouse/day) for either daytime only, nighttime only, or an entire day (24 h) of animals fed HFD2w or HFD-DF2w. (D) Plasma AST or ALT levels and liver/body weight ratio of the animals. (E) Levels of triglycerides, cholesterol, and glucose levels in the animal plasma samples. ∗, ∗∗, and ∗∗∗ indicate p ≤ 0.05, p ≤ 0.01, and p ≤ 0.001, respectively. ns indicates non-significance. Data are represented as mean ± SD. The data shown represent three isolated experiments. See also Figure S3.

• 

  Figure 7. Daytime feeding or apelin treatment could influence TG mice for NAFLD features (A–D) Liver tissues of mice fed HFD2w+Saline (A, C) or HFD2w+Apelin (B, D) were processed for H&E staining, and representative images were shown (A, B), or for whole tissue extracts before immunoblots for the indicated molecules (C, D). Yellow arrows indicate infiltrated immune cells, and red arrowheads indicate damaged hepatocytes. Note the major fat droplet accumulation in TG mice and minor accumulation in WT mice. Scale of 100 μm. (E) SNU449-EV and SNU449-TM4SF5 cells were treated with apelin and/or palmitic acid for 24 h before harvests and immunoblots. The data shown represent three isolated experiments.

• 

  Figure 8. Human NAFLD patients increased apelin as NAFLD developed (A) TM4SF5-related molecules and pathological features in 9 sample pairs of liver tissue and serum from non-NAFLD, NASH-fibrosis, or NASH-Cirrhosis patients. (B) Correlation between TM4SF5 expression (band intensity normalized to β-actin band intensity) in liver tissues and serum apelin levels in the 9 sample pairs (p = 0.1746). (C–H) Correlation between TM4SF5 expression (band intensity normalized to β-actin band intensity) in liver tissues (C), serum apelin (D), insulin (E), glucose (F), triglycerides (G), and total cholesterol (H) levels in the 7 sample pairs (2 non-NAFLD, 4 NASH-Fibrosis, and 1 NASH-Cirrhosis sample pairs). (I and J) Correlation between serum apelin levels and NAS (E) or BMI (F) in the 7 sample pairs (p = 0.0522 or 0.1447, respectively). The unpaired two-tailed Student’s t test was performed. Data are represented as mean ± SD. (K) Working model for hepatocyte TM4SF5-mediated abnormal food intake and apelin expression, leading to DNL, a pro-inflammatory environment, and peroxisomal β-oxidation, which inhibit lipophagy during the initial stages of NAFLD development. See also Figure S4.