Table 1.
Primers for real-time PCR in mouse.
Fig 1.
Effect of toyocamycin (TM) on palmitic acid (PA)-induced X-box binding protein-1 (XBP-1) splicing and apoptosis in cultured hepatocytes.
(A) Huh-7 cells were treated with vehicle, TM (1 μM), PA (800 μM), or PA + TM for 8 h. Left upper panel: XBP-1 cDNA was amplified by real-time polymerase chain reaction (PCR) and incubated with PstI for 1 h. Non-spliced XBP-1 showed 189-bp and 285-bp products, whereas spliced XBP-1 showed a 448-bp product. Left upper panel: The histogram shows quantification of the spliced XBP-1 mRNA. Right upper panel: Total XBP-1 mRNA was assessed by real-time PCR. The data are shown as fold changes relative to the values for the control group. Right lower panel: Huh-7 cells were treated with vehicle, TM (1 μM), PA (800 μM), or PA + TM for 4, 8, and 16 h. Spliced XBP-1 was assessed by immunoblotting. The data shown represent the results from three experiments. (B) Huh-7 cells were treated with PA and TM (1 or 3 μM) for 24 h. Rat primary cells were incubated with or without PA (800 μM) and TM (0.5 μM) for 12 h. (C) A caspase-3/7 assay was performed to biochemically confirm apoptosis. The data are shown as fold changes relative to the values for the control group. All data are expressed as mean ± standard error (n = 3, * indicates P < 0.05).
Fig 2.
Effect of toyocamycin (TM) on free fatty acid-mediated steatosis.
Vehicle-treated cells were used as controls. Huh-7 cells and primary rat cells were treated with TM (1 μM), palmitic acid (PA, 200 μM) + oleic acid (OA, 200 μM), or PA + OA + TM for 24 h. Nile red staining was performed to assess cellular steatosis. Red fluorescence shows lipids and blue fluorescence shows 4′,6-diamidino-2′-phenylindole dihydrochloride-stained sections. Signals were analyzed at a 400-fold magnification.
Fig 3.
Effect of toyocamycin (TM) on the expressions of Bax-dependent Bim, DR5, and CHOP and JNK phosphorylation.
(A and B) Vehicle-treated cells were used as controls. Huh-7 cells were treated with vehicle, TM (1 μM), PA (800 μM), or PA + TM for 8 h. (C) CHOP and DR5 mRNA levels were quantified by real-time polymerase chain reaction. The data are shown as fold changes relative to the values for the control group. The level of Bim was evaluated by western blot analysis. (D) Activation of Bax was examined by immunofluorescence using mouse monoclonal 6A7 Bax antibody. Green fluorescence shows activated Bax and blue fluorescence shows nucleic acids identified by 4′,6-diamidino-2′-phenylindole dihydrochloride staining. 6A7-Immunoreactive cells were quantified in 5 random × 40 objective fields for each condition. All data are expressed as mean ± standard error (n = 3). * indicates P < 0.05. (E) The level of total-phospho-JNK was evaluated by western blot analysis. CHOP: cytosine-cytosine-adenosine-adenosine-thymidine enhancer-binding protein homologous protein; DR5: death receptor; JNK: c-Jun N-terminal kinase.
Fig 4.
Effect of toyocamycin (TM) on the weights of saturated fat, fructose, and cholesterol (FFC) diet-fed mice.
Mice were fed either normal chow or FFC diet for 4 months. Each diet group was divided into TM and saline groups. Mice in the TM group received 0.25 mg/kg/day TM twice weekly for 2 weeks. (A) Weight changes over time in mice fed standard chow (control) or FFC-diet (B) The graph shows food intake before and after TM administration.
Fig 5.
Effect of toyocamycin (TM) treatment on hepatic steatosis in mice fed a saturated fat, fructose, and cholesterol (FFC) diet.
(A) Hematoxylin and eosin (original magnification, 200×) and (B) oil red O-stained sections of liver tissues (original magnification, 400×) from standard chow (control)- and FFC diet-fed mice, with and without TM treatment. The images were captured and analyzed using a fluorescence microscope (BZ-X700, Keyence). The data shown represent the results from three fields imaged from four animals from each treatment group.
Fig 6.
Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (T-cho), triglycerides (TGs), glucose (Glu), and HOMA-IR after toyocamycin (TM) administration.
Mice were fed a standard chow or a saturated fat, fructose, and cholesterol (FFC) diet and administered saline or TM. Plasma AST, ALT, T-chol, TG, and Glu levels were then measured. HOMA-IR was also calculated. All the data are expressed as mean ± standard error (n = 3). * indicates P<0.05.
Fig 7.
Effect of toyocamycin (TM) on the expression of lipogenic genes in mice.
Mice were fed a standard chow or a saturated fat, fructose, and cholesterol (FFC) diet and administered saline or TM. FASN, SCD1, ACOX1, DGAT1, DGAT2, and SREBP1 mRNA expression levels were then measured by real-time polymerase chain reaction. Fold induction was determined by normalization to 18S. All the data are expressed as mean ± standard error (n = 3). * indicates P < 0.05. ACOX1: peroxisomal acyl-coenzyme A oxidase 1; DGAT: diglyceride acyltransferase; FASN: fatty acid synthase; SCD1: stearoyl-CoA desaturase-1; SREBP1: sterol regulatory element-binding protein 1; 18S: 18S rRNA.