A novel 11β-HSD1 inhibitor improves diabesity and osteoblast differentiation

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   Figure 1

   Anti-diabetic effect of KR-67500 on glucose tolerance and insulin sensitivity in DIO-C57BL/6 mice. DIO-C57BL/6 mice were administered with vehicle and KR-67500 (50 mg/kg BW) daily by oral gavage for 28 days. (A) The oral glucose tolerance test. (B) The insulin tolerance test. (closed circle) DIO-C57BL/6 – vehicle, (closed square) DIO-C57BL/6 – KR-67500. (C) Plasma fasting insulin concentration. (D) HOMA-IR index analysis. (E) Ex vivo 11β-HSD1 enzyme activity in liver, inguinal fat, and reproductive fat of KR-67500 treated DIO-C57BL/6 mice. The ex vivo assay was carried out at 16 h after the last administration in the in vivo DIO-C57BL/6 mice study. Results are expressed as mean±s.e.m. for n=6–9 mice per group. *P<0.05, ***P<0.001 vs DIO-C57BL/6 vehicle group.
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   Figure 2

   Anti-adipogenic effect of KR-67500 on lipid accumulation (A) and adipogenic markers expression (B) in cortisone-treated 3T3-L1 cells. Preadipocytes (control) were induced to differentiate with 20 μg/ml insulin, 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, 1 μM cortisone, and KR-67500 (10 and 20 μM), or CBX (20 μM) for 3 days, which was then replaced with medium containing 1 μM cortisone, 20 μg/ml insulin, and compounds for 2 days, and the cells were then cultured for 1 day in culture medium. Values are mean±s.e.m. of data from two separate experiments with three replicates. ^###P<0.001 vs control group; *P<0.05, **P<0.01, ***P<0.001 vs vehicle group. A full colour version of this figure is available via http://dx.doi.org/10.1530/JME-13-0177.
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   Figure 3

   The 11β-HSD1 enzyme activity of KR-67500 in mouse 3T3-L1 adipocytes (A) and in the inguinal or reproductive fat of C57BL/6 mice (B). (A) In vitro 11β-HSD1 enzyme inhibitory activity and expression of KR-67500 in adipocytes. The 11β-HSD1 expression was determined by real-time PCR. Values are mean±s.e.m. of data from two separate experiments with three replicates. ^##P<0.01 vs preadipocyte group; *P<0.05 vs cortisone-treated group. (B) Ex vivo concentration dependency assay on 11β-HSD1 enzyme inhibitory activity of KR-67500. Male C57BL/6 mice were orally gavaged with vehicle or KR-67500 and killed 2 h post dose. (C) Ex vivo time-dependency assay on 11β-HSD1 inhibitory activity of KR-67500. Male lean mice were orally gavaged with vehicle or 10 mg/kg KR-67500 and killed 2 and 6 h post dose. Results are expressed as mean±s.e.m. for n=4 mice per group. *P<0.05, **P<0.01, ***P<0.001 vs C57BL/6 vehicle group.
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   Figure 4

   Osteogenic activity of KR-67500. Osteogenic activity of KR-67500 was evaluated by visualizing the expression of ALP (A) and measuring the mRNA expressions of ALP and osteogenic BMPs in C2C12 cells (B). Real-time PCR and ALP staining were carried out on differentiation days 3 and 6 respectively. Cells were incubated with vehicle (DMSO) or KR-67500 (10 μM) in the presence of BMP2 for mRNA analysis. ^#P<0.05, ^###P<0.001 vs control group; *P<0.05, ***P<0.001 vs BMP2-treated group. A full colour version of this figure is available via http://dx.doi.org/10.1530/JME-13-0177.
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   Figure 5

   Inhibitory effect of KR-67500 on the activity (A) and the expression of 11β-HSD1 in differentiated C2C12 cells (B). (A) Mouse-differentiated C2C12 cells were seeded at 2×10⁴ cells/well onto 96-well plates and incubated in a medium containing 160 nM cortisone in the presence or absence of compounds for 48 h at 37 °C. Results are expressed as mean±s.e.m. of triplicate experiments. ***P<0.001 vs cortisone-treated group. (B) Effect of KR-67500 on mRNA 11β-HSD1 expression in BMP2-treated C2C12 cells. The mRNA 11β-HSD1 expression was determined by real-time PCR. Values are mean±s.e.m. of data from two separate experiments with three replicates. ^###P<0.001 vs control group; *P<0.05 vs BMP2-treated group.
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   Figure 6

   Anti-osteoclastogenic activity of KR-67500. BMM cells were cultured for 4 days in the presence of RANKL (5 ng/ml) and M-CSF (30 ng/ml) with KR-67500 or CBX. Multinucleated osteoclasts were visualized to red-colored giant cells by TRAP staining (A) and their activity was also measured (B). (C) Effect of KR-67500 on osteoclastogenesis-related gene expressions. After treating the vehicle (DMSO) or KR-67500 (5 μM) for 1 h, BMMs were treated with RANKL (5 ng/ml) for the indicated number of days and then mRNA expression levels were analyzed by real-time PCR. *P<0.05, **P<0.01, ***P<0.001 vs control group. A full colour version of this figure is available via http://dx.doi.org/10.1530/JME-13-0177.

• © 2014 Society for Endocrinology