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Thiazolidinedione (Pioglitazone) Blocks P. gingivalis- and F. nucleatum, but not E. coli, Lipopolysaccharide (LPS)-induced Interleukin-6 (IL-6) Production in Adipocytes

¹ Department of Patho-physiology/Periodontal Science, and
² Oral Microbiology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8525, Japan;

View larger version (77K): [in a new window]   Figure 1. The effect of pioglitazone on the morphology of mouse 3T3-L1 adipocytes. Mouse 3T3-L1 adipocytes were differentiated into mature adipocytes as in MATERIALS & METHODS. We confirmed maturation by staining the cells with oil red O (A: right panel). When more than 95% of the cells were stained with oil red O, similarly prepared cells were co-incubated with indicated concentrations of pioglitazone. Twenty-four hrs later, the cells were stained with oil red O, and the morphology of the cells was observed by microscopy. Pioglitazone changed adipocyte appearance from large to small size (B). For comparison, the diameters of 10 randomly selected oil-red-positive cells per high-power field (400x) were measured and compared (C). The data were expressed as % diameter of the cells against the mean diameter (N = 10) of control (untreated) cells. The mean diameter of adipocytes significantly decreased by 1 µM and 10 µM of pioglitazone (100.0 ± 11.3% for pioglitazone-untreated cells, 61.7 ± 17.7% for 1 µM pioglitazone-treated cells, 54.3 ± 18.8% for 10 µM pioglitazone-treated cells, N = 10 for all groups). *p < 0.01, compared with the cells without pioglitazone.

View larger version (19K): [in a new window]   Figure 2. The effects of E. coli LPS on IL-6 production, and of pioglitazone on E. coli LPS-induced IL-6 production in adipocytes. Mouse 3T3-L1 adipocytes were differentiated in 24-well tissue culture plates as described in MATERIALS & METHODS. After confirming the maturation of adipocity by staining the cells with oil red O, we stimulated the cells with indicated concentrations of E. coli LPS. In some cultures, the cells were co-incubated with 10 µM of pioglitazone. Twenty-four hrs later, the cell-culture supernatants were harvested, and the IL-6 concentration was measured by ELISA. All experiments were done in triplicate, and statistical differences were calculated by Student’s t test. In pioglitazone-treated cells, IL-6 production was compared with that of untreated cells when stimulated with the identical concentration of E. coli LPS. Mean IL-6 concentration ± standard deviation in each culture condition was calculated (1094.2 ± 68.6 pg/mL for LPS-unstimulated cells, 3189.7 ± 77.7 for 1 ng/mL of LPS-stimulated cells, 3271.5 ± 65.0 pg/mL for 10 ng/mL of LPS-stimulated cells, 3353.3 ± 53.2 pg/mL for 100 ng/mL of LPS-stimulated cells, 365.1 ± 5.0 pg/mL for LPS-unstimulated cells co-incubated with pioglitazone, 2087.4 ± 250.0 pg/mL for 1 ng/mL of LPS-stimulated cells co-incubated with pioglitazone, 2181.5 ± 16.0 pg/mL for 10 ng/mL of LPS-stimulated cells co-incubated with pioglitazone, and 2331.9 ± 11.8 pg/mL for 100 ng/mL of LPS-stimulated cells co-incubated with pioglitazone, N = 3 for all groups). *p < 0.005 and **p < 0.0001, compared with the cells stimulated with the same concentration of LPS in the absence of pioglitazone.

View larger version (16K): [in a new window]   Figure 3. The effects of P. gingivalis LPS on IL-6 production, and of pioglitazone on P. gingivalis LPS-induced IL-6 production in adipocytes. Mouse 3T3-L1 adipocytes were differentiated in 24-well tissue culture plates as described in MATERIALS & METHODS. After confirming maturation of adipocity by staining the cells with oil red O, we stimulated the cells with indicated concentrations of P. gingivalis LPS. In some cultures, the cells were co-incubated with 10 µM of pioglitazone. Twenty-four hrs later, the cell-culture supernatants were harvested, and the IL-6 concentration was measured by ELISA. All experiments were done in triplicate, and statistical differences were calculated by Student’s t test. In pioglitazone-treated cells, the IL-6 production was compared with that of untreated cells when stimulated with the identical concentration of P. gingivalis LPS. Mean IL-6 concentration ± standard deviation in each culture condition was calculated (1094.2 ± 68.6 pg/mL for LPS-unstimulated cells, 1097.0 ± 13.2 pg/mL for 1 ng/mL of LPS-stimulated cells, 1796.9 ± 57.7 pg/mL for 10 ng/mL of LPS-stimulated cells, 1794.7 ± 8.2 pg/mL for 100 ng/mL of LPS-stimulated cells, 365.1 ± 5.0 pg/mL for LPS-unstimulated cells co-incubated with pioglitazone, 299.2 ± 1.8 pg/mL for 1 ng/mL of LPS-stimulated cells co-incubated with pioglitazone, 267.9 ± 4.1 pg/mL for 10 ng/mL of LPS-stimulated cells co-incubated with pioglitazone, and 372.4 ± 9.5 pg/mL for 100 ng/mL of LPS-stimulated cells co-incubated with pioglitazone, N = 3 for all groups). **p < 0.0001, compared with the cells stimulated with the same concentration of LPS in the absence of pioglitazone.

View larger version (15K): [in a new window]   Figure 4. The effects of F. nucleatum LPS on IL-6 production, and of pioglitazone on F. nucleatum LPS-induced IL-6 production in adipocytes. Mouse 3T3-L1 adipocytes were differentiated in 24-well tissue culture plates as described in MATERIALS & METHODS. After confirming maturation of adipocity by staining the cells with oil red O, we stimulated the cells with indicated concentrations of F. nucleatum LPS. In some cultures, the cells were co-incubated with 10 µM of pioglitazone. Twenty-four hrs later, the cell-culture supernatants were harvested, and the IL-6 concentration was measured by ELISA. All experiments were done in triplicate, and the statistical differences were calculated by Student’s t test. In pioglitazone-treated cells, the IL-6 production was compared with that of untreated cells when stimulated with the identical concentration of F. nucleatum LPS. Mean IL-6 concentration ± standard deviation in each culture condition was calculated (1094.2 ± 68.6 pg/mL for LPS-unstimulated cells, 1308.3 ± 33.6 pg/mL for 1 ng/mL of LPS-stimulated cells, 1283.8 ± 6.4 pg/mL for 10 ng/mL of LPS-stimulated cells, 1552.9 ± 42.7 pg/mL for 100 ng/mL of LPS-stimulated cells, 365.1 ± 5.0 pg/mL for LPS-unstimulated cells co-incubated with pioglitazone, 223.7 ± 11.8 pg/mL for 1 ng/mL of LPS-stimulated cells co-incubated with pioglitazone, 236.0 ± 5.9 pg/mL for 10 ng/mL of LPS-stimulated cells co-incubated with pioglitazone, and 383.8 ± 5.5 pg/mL for 100 ng/mL of LPS-stimulated cells co-incubated with pioglitazone, N = 3 for all groups). **p < 0.0001, compared with the cells stimulated with the same concentration of LPS in the absence of pioglitazone.