arteriolecorre- lations between morphology and function. Nephrol Dial Transplant 21: 2703707, 2006. 32. Rosivall L, Mirzahosseini S, Toma I, Sipos A, Peti-Peterdi J. Fluid w in the juxtaglomerular interstitium visualized in vivo. Am J Physiol Renal Physiol 291: F12411247, 2006. AJP-Cell Physiol doi:10.1152/ajpcell.00138.2011 ajpcell Downloaded from ajpcell.physiology on March 6, 2012 Page 10 Adiponectin and Hippocampal Decitabine Neurogenesis adult neurogenesis, molecular and cellular mechanisms that control proliferation, differentiation, and self-renewal of neural stem cells in the adult hippocampus remain unclear (37).
We and others have previously reported that adipokines, such as leptin and TNF- , regulate adult hippocampal neurogenesis, with the former increasing neurogenesis and the latter inhibit- ing neurogenesis (38 42). However, the role of adiponectin in adult Sorafenib hippocampal neurogenesis has not been identified. In the present study, we examined the expression of adiponectin receptors in adult hippocampal neural stem/progenitor cells (hNSCs) and investigated the effects of adiponectin on prolif- eration, apoptosis, and differentiation of adult hNSCs. Further- more, we explored the intracellular signal transduction path- ways that mediate the effect of adiponectin on adult hNSCs.
EXPERIMENTAL PROCEDURES Cell Culture dult hNSCs were isolated from the hip- pocampus of adult Fisher rats and were positive for stem cell markers and negative for neuronal and astrocyte lineage mark- ers (Millipore). The hNSCs were propagated in neural expan- sion medium (DMEM/F-12 nutrient mix, B-27 supplement, 2 m M L purchase OSI-420 -glutamine, 100 units/liter penicillin, and 20 ng/ml FGF-2) on poly- L -ornithine/laminin-coated plates. For differentiation, cells were maintained in FGF-2-free neural expansion medium with the addition of 1.0 M retinoic acid and 0.5% fetal bovine serum (FBS) to induce mixed neuronal-glial differentiation for 6 days (43). The purity of hNSCs was accessed by immunocy- tochemical staining with specific neuronal stem cell marker nestin. Unless indicated otherwise, cells were plated at a density of 2 10 4 cells/cm 2 in all experiments.
Cell Proliferation Assay he MTT assay was used to deter- mine cell proliferation. Cells were plated in 96-well plates and grown overnight, followed by treatment with various concen- trations (0 g/ml) of recombinant globular adiponectin (Phoenix Pharmaceuticals Inc., Belmont, CA) or full-length adiponectin (R&D Systems, Minneapolis, MN). The MTT stock order OSI-420 solution (5 mg/ml; Sigma-Aldrich) was added to the cells and incubated at 37 for 2 h. Then supernatant was carefully removed and replaced with 100 l of DMSO for 10 min. The absorbance at 590 nm was measured with a SpectraMax 340 microplate reader (Molecular Devices, Sunnyvale, CA). To determine whether inhibition of AMPK or p38MAPK affects cell proliferation, cells were pretreated with different doses of the AMPK inhibitor Compound C (0.02.0 M ; EMD Chem- icals Inc., Gibbstown, NJ) or p38MAPK inhibitor SB203580 To determine the expression of adiponectin receptors in hNSCs, a classical Chinese medicine combination of rabbit anti-AdipoR1 or anti-AdipoR2 (1:1000; a gift from Dr. Feng Liu, University of Texas Health Science Center, San Antonio, TX) and mouse anti-nestin anti- bodies (1:1000; Millipore) were utilized. To determine the fate of differentiated stem cells, anti-Tuj1 (1:500; Covance, Prince- ton, NJ) and anti-GFAP antibodies (1:1000; Millipore) were used. Following incubation with primary antibodies overnight, cells were rinsed with 0.1 M PBS and incubated with secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 546 (1:400; Invitrogen) for 4 h. The chamber slides were then cov- erslipped using ProLong Gold Antifade reagent containing 4 ,6-diamidino-2-phenylindole (DAPI) (Invitrogen) for nucleariol 195: 40210, 2003. 9. Dvorak AM, Feng D. The vesiculo-vacuolar organelle (VVO). A new endothelial cell permeability organelle. J Histochem Cytochem 49: 419 432, 2001. 10.