Table 1.
Primer sequences used in this study.
Fig 1.
Inhibitory effect of spiramycin on lipid accumulation in 3T3-L1 adipocytes.
(A) The effect of spiramycin on lipid droplet formation was measured by Oil Red O staining. 3T3-L1 preadipocytes were differentiated into adipocytes in the presence of various concentrations of spiramycin (2.5–20 μM) for 6 days. Representative cell images were captured at 200× magnification. GM, growth media. DM, differentiation media. (B) Quantification of intracellular lipid accumulation. Oil Red O stained lipids were extracted in absolute isopropanol, after which the absorbance of the solution was measured at 510 nm. (C) Quantification of triglyceride content. The bar graphs show the mean ± S.D. of 3 independent experiments (*p< 0.05, **p< 0.01, and ***p< 0.001 compared with the non-spiramycin-treated DM control).
Fig 2.
Inhibitory effect of spiramycin on expression of adipogenic transcriptional factors and their adipocyte-specific target genes in 3T3-L1 adipocytes.
(A) 3T3-L1 preadipocytes were differentiated into adipocytes in the presence of spiramycin for 6 days. The transcript levels of major adipogenic transcription factors (PPARγ, C/EBPα, and SREBP1) and their adipocyte-specific target genes (aP2, GLUT4, and FAS) were evaluated by qRT-PCR. (B) Western blotting analysis showing the effect of spiramycin on protein levels of major adipogenic transcription factors (PPARγ, C/EBPα, and SREBP1). The numbers at the bottom of the figure indicate the relative intensity of each band (fold-change in comparison with that of the control group), which was estimated using Multi Gauge software version 3.0.
Fig 3.
Effect of spiramycin on phosphorylation of AMPK and ACC during 3T3-L1 adipocyte differentiation.
Confluent 3T3-L1 preadipocytes (Day 0) were incubated into DM including MDI with specified concentrations of spiramycin. After 1 h, protein levels of phosphorylated AMPK and ACC (p-AMPK and p-ACC) were analyzed by western blotting. The numbers at the bottom of the figure indicate the relative band intensity normalized to that of the non-phosphorylated protein (fold-change in comparison with that of the control group).
Table 2.
The changes of body and organ weight, and food intake.
Fig 4.
Effects of spiramycin on HFD-induced obesity in C57BL/6 mice.
Spiramycin (25 or 50 mg/kg) or orlistat (50 mg/kg) were administered by oral gavage for 8 weeks while the mice were fed the HFD. (A) Experimental outline. (B) Body weight was measured 3 times per week. The group that received HFD alone(■) showed steady body weight gain, while the spiramycin-(▲ or ▼) and orlistat-treated(◆) groups showed significantly attenuated body weight gain. (C) Adipose tissue weight in subcutaneous, epididymal, and mesentery fat. (D) Amount of leptin in plasma measured by ELISA. (E) Liver weight. (F) Plasma levels of GOT and GPT measured using a chemical analyzer. (G) H&E-stained images of liver and subcutaneous adipose tissue samples from the normal diet (control), vehicle-treated, spiramycin-treated, and orlistat-treated groups. The results are expressed as the mean ± SD for each group (n = 8).
Table 3.
Effects of spiramycin on GOT, GPT, and serum lipid levels in mice fed different experimental diets for 8 weeks.