Elevated TTR is associated with the development of NAFLD.

(A) Quantitative real-time PCR (qRT-PCR) detected Ttr mRNA levels from liver sections in NASH patients and non-NAFLD controls (n = 5 per group). (B-E) TTR was overexpressed in ND or HFD-fed mice by tail vein injection of adenovirus (n = 4 per group). (B) Western blot analysis of hepatic protein level of TTR. (C) Serum TTR levels were detected by ELISA. Hepatic TG contents (D) and Oil Red O staining (E) in mouse liver tissues. Scale bar: 100 μm. (F-H) TTR was overexpressed in AML12 cells by adenovirus. (F) Western blot analysis of protein level of TTR-FLAG. Hepatic TG contents (G) and Oil Red O staining (H) in AML12 cells. Scale bar: 100 μm. Data are shown as the mean ± SEM. P values were from 2-tail unpaired t test (A) and 1-way ANOVA (B, C, D and G). #P = 0.057, *P < 0.05, **P < 0.01, ***P < 0.001. ND, normal diet; HFD, high fat diet; Ad-Ttr, Adenovirus-Ttr; NS, no significance; TTR-OE, TTR overexpression; TG, triglyceride.

The effect of TTR-KD on insulin resistance.

(A-F) TTR knockdown was achieved in the HFD-fed mice as mentioned in the Materials and Methods section (n = 7-8 per group). (A) qRT-PCR detected hepatic Ttr mRNA levels in HFD-fed mice. Serum TTR levels were examined by western blot (B) and ELISA (C). (D) The body weight of HFD-fed mice was recorded per week for 19 weeks. Blood glucose levels and AUC of IPGTT (E) and ITT (F) in HFD-fed mice. (G-L) TTR knockdown was achieved in the GAN diet-fed mice as described in the Materials and Methods section (n = 5–6 per group). (G) qRT-PCR detected hepatic Ttr mRNA levels in GAN diet-fed mice. Serum TTR levels were measured by western blot (H) and ELISA (I). (J) The body weight of GAN diet-fed mice was recorded per week for 17 weeks. Blood glucose levels and AUC of OGTT (K) and ITT (L) in GAN diet-fed mice. Data are shown as the mean ± SEM. P values were from 1-way ANOVA (A, C, D, J, E, F, G, I, K and L). #P = 0.07, *P < 0.05, **P < 0.01, ***P < 0.001. TTR-KD, TTR knockdown; GAN, Gubra-Amylin NASH; IPGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test; OGTT, oral glucose tolerance test; AUC, area under curve.

TTR knockdown improved liver lipid deposition, liver function as well as inflammatory gene expression in HFD-fed mice.

Liver/body weight ratio (A) and hepatic TG contents (B) were measured in the TTR-KD and control mice. (C) Representative images of Oil Red O and H&E staining of liver tissue; scale bar: 100 μm. (D) Liver steatosis, lobular inflammation and NAFLD activity scores in HFD-fed mice. Liver function evaluated by serum ALT, AST levels (E) and lipid levels (F). (G) Related gene expressions of liver inflammation and fibrosis. (H) Representative images of Masson’s trichome and Sirius red staining of liver sections; scale bar: 100 μm. n = 7-8 per group. Data are shown as the mean ± SEM. P values were from 2-tail unpaired t test (D) and 1-way ANOVA (A, B, E, F and G). #P = 0.06, *P < 0.05, **P < 0.01, ***P < 0.001. H&E, hematoxylin and eosin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol.

TTR knockdown improved liver lipid deposition, liver function as well as inflammatory gene expression, but did not significantly alter liver tissue fibrosis in GAN diet-fed mice.

Representative images of general liver appearance (A), liver/body weight ratio (B) and hepatic TG contents (C) in the TTR-KD and control mice, respectively. (D) Representative images of Oil Red O and H&E staining of liver tissue; scale bar: 100 μm. (E) Liver steatosis, lobular inflammation and NAFLD activity scores in GAN diet-fed mice. Liver function evaluated by serum ALT, AST levels (F) and lipid levels (G). (H) Related gene expressions of liver inflammation and fibrosis. (I) Representative images of Masson and Sirius Red staining of liver sections; scale bar: 100 μm. n = 5-6 per group. Data are shown as the mean ± SEM. P values were from 2-tail unpaired t test (E) and 1-way ANOVA (B, C, F, G and H). #P = 0.06, *P < 0.05, **P < 0.01, ***P < 0.001.

TTR knockdown downregulated the expression of ER lipid synthesis-related genes.

(A-C) Bioinformatics analyses for the relationship between TTR knockdown and ER lipid metabolism in the liver of NAFLD mice (n = 3 per group). (A) KEGG pathway enrichment analysis and GO analysis for biological processes, cell components and molecular function regulated by differentially expressed genes (DEGs) (P< 0.05, ≥ 1.0-fold change). (B) Representative heat map of DEGs associated with ER lipid metabolism. (C) Non-targeted lipidomics analysis showed a lipid bar plot in differences of lipid species between TTR-KD mice and control mice fed on HFD. (D-I) qRT-PCR was used to confirm DEGs that were enriched in ER lipid metabolism-related pathways (n = 7-8 per group in HFD mice; n = 5-6 per group in GAN-diet mice). The mRNA expression levels of genes related to de novo lipogenesis (D, G), fatty acid oxidation (E, H) and ER lipid synthesis (F, I) in the livers of TTR-KD mice fed on HFD and GAN diet. Data are shown as the mean ± SEM. P values were from 1-way ANOVA. #P = 0.07, *P < 0.05, **P < 0.01, ***P < 0.001. KEGG, the Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology.

TTR knockdown inhibited ER stress and upregulated SERCA2 expression in the liver of NAFLD mice.

Western blot analysis of the protein levels of p-PERK/p-eIF2α/CHOP and ATF6 signaling pathways in the liver of HFD mice (A) and GAN-diet mice (B). SERCA2 protein expression examined by western blot in the liver of HFD mice (C), GAN-diet mice (D), and FFA-induced AML12 cells transfected with siRNA (E). n = 7-8 per group in HFD mice; n = 5-6 per group in GAN-diet mice. Data are shown as the mean ± SEM. P values were from 1-way ANOVA. #P = 0.06, *P < 0.05, **P < 0.01, ***P < 0.001.

TTR could be “taken up” by hepatocytes and localized close to the ER.

(A) AML12 cells transfected with the mCherry-ER-3 plasmid for 48 hours were then subjected to serum starvation for 6 hours, followed by the addition of 50 μg/mL Alexa Fluor 488 labeling TTR probes and incubated at 37°C in the dark for 30 minutes. After washing off the free probes, live cell imaging was immediately performed by confocal microscope. Alexa Fluor 488 labeling TTR was observed at an excitation wavelength of 488 nm, while the ER fluorescing red was visible at an excitation wavelength of 594 nm; scale bars: 10 μm (up), 5 μm (down). (B) Immunofluorescence staining of TTR and Calnexin in AML12 and HepG2 cells using confocal microscopy. TTR was observed at an excitation wavelength of 594 nm, and Calnexin at 488 nm. Nuclei were stained with DAPI. Scale bar: 5 μm.

The effect of TTR on calcium homeostasis in AML12 cells.

After the treatment of TTR (10 μg/ml) or PBS, the fluorescence intensity of Ca2+change was monitored in Fluo-8 AM-loaded AML12 cells (A) and in AML12 cells with pcDNA-D1ER plasmid transfection (B). 5 μM Tg was added to block calcium transport from the cytosol to the ER. The fluorescence intensity of Ca2+ change was detected in TTR-OE AML12 cells by adenovirus (C) and in TTR-KD stable cell lines (D). AML12 cells were incubated with BSA or FFA for 12 h. (E) Resting cytosolic calcium signals were detected in the TTR-KD stable cell lines by Cal-590 AM loading; scale bar: 10 μm. (F) Statistical chart of resting cytosolic calcium intensity in stable cell lines induced by FFA or BSA. (G) After the treatment of Tg (5 μM), the fluctuation of cytosolic calcium signal was monitored in Cal-590 AM-loaded AML12 cells or TTR-KD stable cell lines induced by FFA or BSA. (H) Statistical chart of the peak release ratio of cytosolic calcium intensity caused by Tg stimulation in stable cell lines induced by FFA or BSA. (I) Relative SERCA2 activity in BSA/FFA treated AML12 cells with Ttr siRNA infection or not. The activity of SERCA was calculated from the hydrolyzed ATP level (nmol) normalized with the protein content (mg) and reaction time (min). Data are shown as the mean ± SEM. P values were from 2-tail unpaired t test (A, B, C and D) and 1-way ANOVA (F, H). n = 10-20 cells per group. *P < 0.05, **P < 0.01, ***P < 0.001. Tg, thapsigargin.

Protein interaction between TTR and SERCA2 in hepatocytes.

(A) Immunofluorescence staining of TTR and SERCA2 in AML12 and HepG2 cells using confocal microscopy; scale bar: 10 μm. (B, C) HEK-293T cells overexpressed TTR-FLAG and SERCA2-MYC, and Co-IP was used to detect the protein interaction. (B) TTR and SERCA2 shared a common murine genetic origin. (C) TTR and SERCA2 shared a common human genetic origin. (D) AML12 cells overexpressed FLAG-TTR, and CoIP was used to detect the interaction between TTR-FLAG protein and endogenous SERCA2 protein. TTR-FLAG, TTR protein labeled with FLAG; SERCA2-MYC, SERCA2 protein labeled with MYC; Co-IP, co-immunoprecipitation.