Journal: Molecular Endocrinology

Article Title: 11β-HSD1 Modulates LPS-Induced Innate Immune Responses in Adipocytes by Altering Expression of PTEN

doi: 10.1210/me.2014-1287

Figure Lengend Snippet: A, Emodin dose dependently inhibits 11β-HSD1 activity in 3T3-L1 adipocytes. Cells were treated with 10-ng/mL LPS in the presence or absence of emodin at the indicated concentrations for 24 hours, followed by incubation with 100μM reduced nicotinamide adenine dinucleotide phosphate and 1μM cortisone. Cortisol production in the supernatant was assayed using a Cortisol EIA kit. Cortisol was undetectable in the absence of exogenous cortisone, so cortisol production reflects conversion of exogenous cortisone to cortisol by 11β-HSD1 activity. B, Emodin inhibits IL-6, IL-1β, and TNF-α transcripts in 10-ng/mL LPS-treated 3T3-L1 cells. LPS-treated 3T3-L1 cells were incubated with vehicle or 10μM emodin for 24 hours. The levels of mRNA were measured by qRT-PCR and normalized to18S RNA. C, Emodin inhibits LPS or palmitate-induced IL-6 production but not the basal level. Left panel, Cells were treated with vehicle or 10-ng/mL LPS in the absence (■) or presence (□) of 10μM emodin. Right panel, Cells were incubated with vehicle or 10μM emodin in the presence of 250μM palmitate. After 24 hours, IL-6 secretion was assayed by ELISA. The data are mean ± SEM and are expressed as fold-changes relative to basal production of IL-6 (left panel) or control (right panel). D, The inhibitory effects are shown of 10μM emodin, 5nM 11β-HSD1-targeted siRNA, or a combination of both, on IL-6 production. An unrelated control RNAi had no significant effect on IL-6 production. Cells were transfected with 11β-HSD1-targeted siRNA or a control RNAi (control) in the presence or absence of emodin for 24 hours, and IL-6 production was measured by ELISA. E, Cortisone induces IL-6 production and that is reversed by emodin. LPS-treated cells were incubated with vehicle (control), or 10μM emodin, or cortisone (1pM) with or without 10μM emodin. After 24 hours, IL-6 production was assayed by ELISA. Unless otherwise specified, all values are mean ± SEM and are expressed as fold-changes relative to their respective controls. All results are from 3 independent experiments each performed in duplicate. *, .01 < P < .05; **, P < .01.

Article Snippet: After 3 hours, cell culture medium was collected, and cortisol production was assayed using a Cortisol EIA kit (Enzo Life Sciences), as per the manufacturer's instructions.

Techniques: Activity Assay, Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection