Fig 1.
Expression of adiponectin receptors in human sebaceous glands and sebocytes.
(A) Human sebaceous glands were assayed for adiponectin receptor (AdipoR)1 and AdipoR2 by immunohistochemistry. Inset, isotype control. (B) Immunofluorescence labeling of AdipoR1 and AdipoR2 (green) in human sebocytes. Nuclei were counterstained with DAPI (blue). Inset, isotype control. (C, D) Western blotting and RT-PCR of sebocyte lysates. Human keratinocytes and fibroblasts expressing AdipoR1 and AdipoR2 were used as positive controls. Scale bars = 20 μm. Sebo, sebocytes; KC, keratinocytes; FB, fibroblasts.
Fig 2.
Effects of adiponectin on cell proliferation in human sebocytes.
(A) The effects of adiponectin on sebocyte viability were examined by MTT assay. (B) Rate of [3H]-thymidine incorporation into sebocytes treated with various doses of adiponectin, calculated as a percentage of the value in untreated cells. (c) Dose-dependent effects of adiponectin on sebocyte proliferation were examined using microscopy from low cell seeding assays. Scale bars = 400 μm. Data represent means ± SEM (n = 6). Data were analyzed using Student’s t test (*P < 0.05).
Fig 3.
Effects of adiponectin on lipid production in human sebocytes.
(A) Detection of intracellular lipids in sebocytes treated with adiponectin using microscopy after Oil Red O and Nile red staining. Scale bars = 20 μm. (B) Lipid levels in sebocytes treated with various doses of adiponectin, calculated as percentages of the value of untreated cells. (C) Relative abundance of major lipid classes determined by thin-layer chromatography. Human sebocytes grown in the presence of [14C]-acetate after treatment with adiponectin, and changes in specific lipid components such as cholesterol, triglyceride, wax ester, and squalene, were analyzed. (D) The effect of adiponectin in three-dimensional (3D) culture of human sebocytes. Sebocytes in 400 μL of medium were added to the matrigel layer (50–100 μL) adding to glass-bottom dishes. Sebocytes were treated with 200 ng/mL adiponectin and the overlay medium was replaced every 2 days during culture of the cells for 7 days. Organoid structures in 3D culture of sebocytes were stained with hematoxylin and eosin (H&E), Nile red, SREBP and epithelial membrane antigen (EMA). Scale bars = 20 μm. Data represent means ± SEM (n = 8). Data were analyzed using Student’s t test (*P < 0.05, **P < 0.01).
Fig 4.
Induction of lipid synthesis by adiponectin in human sebocytes by activating APPL1-AMP activated protein kinase (AMPK) signaling.
(A) After sebocytes were treated with adiponectin, whole cell lysates were prepared and analyzed by Western blotting. Blots were incubated with specific antibodies. Densitometric analyses of these protein signals were normalized relative to those for actin controls. (B) Sebocytes were transfected with control, AdipoR1 or AdipoR2-targeting siRNA and the intracellular protein levels of each signals were determined by Western blotting. (C) Sebocytes were incubated with different concentrations of compound C and protein signals were determined by Western blotting and intracellular lipid levels were measured by Oil Red O staining. Data represent means ± SEM (n = 6). Data were analyzed by Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001 vs. untreated cells; †P < 0.05, ‡P < 0.01 vs. adiponectin-treated cells).