Effect of serum on differentiation of porcine adipose stromal-vascular cells in primary culture

Hong

Journal of Biological Chemistry

et al. 2003

118

Leptin, the Ob gene product, has emerged recently as a key regulator of bone mass. However, the mechanism mediating leptin effect remains controversial. Because the action of leptin is dependent on its receptors, we analyzed their expression in osteoblast-lineage primary human bone marrow stromal cells (hBMSC). Both the short and long forms of leptin receptors were detected in hBMSC. Leptin significantly decreased the viability of hBMSC. This cytotoxic effect was prevented by ZVal-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor, implicating that leptin-induced hBMSC death was caspase-dependent. Further investigation demonstrated that leptin activated caspase-3 and caspase-9, but not caspase-8, and increased the cleavage of poly-(ADP-ribose) polymerase and cytochrome c release into cytosol. Leptin activated ERK, but not p38 and JNK, and up-regulated cPLA2 activity; the latter was abolished by pre-treatment of cells with the MEK inhibitor (PD98059 or U0126) or cPLA2 inhibitor (AACOCF3). PD98059, U0126, and AACOCF3 also diminished the leptin-induced cytochrome c release into cytosol, cell death, and caspase-3 activation. These data indicated that leptin induced hBMSC apoptosis via ERK/cPLA2/cytochrome c pathway with activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase. To our knowledge, this is the first study demonstrating the direct detrimental effect of leptin on bone cells.Leptin, the protein product encoded by obese gene, is a circulating hormone produced primarily by the adipose tissue and is a multifunctional hormone that plays important roles in body weight homeostasis, neuroendocrine function, fertility, immune function, and angiogenesis (1-3). The biological actions of leptin on target tissues are carried out through interaction with its specific receptor, Ob-R (4). A hallmark of Ob-R expression is the presence of several receptor variants (Ob-Ra through Ob-Rf) that are generated by alternative splicing; they share the same extracellular domain but differ in the length of the transmembrane/cytoplasmic coding regions (2). The long Ob-Rb subtype (Ob-R L ) appears as the functional, signal-transducing isoform, responsible for the action of leptin. The roles of the shorter Ob-R isoforms (Ob-R S ) remain to be characterized. Although leptin's precise sites of action are not known, its effect is thought largely mediated via hypothalamus. However, the wide expression of Ob-R L throughout the body suggests that leptin may also operate directly in peripheral tissues (5). There is now a significant amount of evidence implicating that leptin is active in the periphery (6).Recently, leptin has emerged as a key element in the regulation of bone mass. However, the mechanism by which leptin acts upon the bone remains unclear. Thomas et al. (7) reported that leptin enhanced the differentiation of hBMSC, 1 and Steppan et al. (8) reported that the femurs of leptindeficient ob/ob mice were shorter than those in the normal mice, and intraperitoneal injection of leptin significantly increas...

Section: Methodsmentioning

confidence: 99%

Leptin Induces Apoptosis via ERK/cPLA2/Cytochrome c Pathway in Human Bone Marrow Stromal Cells

Hong

Journal of Biological Chemistry

et al. 2003

118

The Journal of Nutritional Biochemistry

“…On Day 14, cells were washed with PBS and fixed with Baker's formalin (10 ml 37% formaldehyde, 10 ml of a 10% calcium chloride solution, 80 ml distilled water) for 30 min at room temperature and then stained with Oil Red O and hematoxylin [16]. After staining, cells were mounted with glycerol gelatin and the images of each dish were captured using ImagePro software (MediaCybernetics, Silver Spring, MD, USA).…”

Section: Oil Red O Stainingmentioning

confidence: 99%

Genistein inhibits differentiation of primary human adipocytes

Park

Della‐Fera

Hausman

et al. 2009

Genistein, a major soy isoflavone, has been reported to exhibit antiadipogenic and proapoptotic potential in vivo and in vitro. It is also a phytoestrogen which has high affinity to estrogen receptor β. In this study, we determined the effect of genistein on adipogenesis and estrogen receptor (ER) α and β expression during differentiation in primary human preadipocytes. Genistein inhibited lipid accumulation in a dose-dependent manner at concentrations of 6.25 μM and higher, with 50 μM genistein inhibiting lipid accumulation almost completely. Low concentrations of genistein (3.25 μM) increased cell viability and higher concentrations (25 and 50 μM) decreased it by 16.48±1.35% (Pb.0001) and 50.68±1.34% (Pb.0001). Oil Red O staining was used to confirm the effects on lipid accumulation. The inhibition of lipid accumulation was associated with inhibition of glycerol-3-phosphate dehydrogenase activity and down-regulation of expression of adipocyte-specific genes, including peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, glycerol-3-phosphate dehydrogenase, adipocyte fatty acid binding protein, fatty acid synthase, sterol regulatory element-binding protein 1, perilipin, leptin, lipoprotein lipase and hormone-sensitive lipase. These effects of genistein during the differentiation period were associated with downregulation of ERα and ERβ expression. This study adds to the elucidation of the molecular pathways involved in the inhibition of adipogenesis by phytoestrogens.

“…As a differentiation inducer, DEX is usually applied either after cell confluence or after seeding in serum for at least 1 day in studies of cell lines (5,13,16) and primary S-V cultures (10,11,19,27). No one has initiated DEX treatment at seeding or before confluence, i.e., during the proliferation phase in porcine S-V cultures.…”

Section: Introductionmentioning

confidence: 99%

Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Zhang

1997

Obesity Research

YU, ZK, JT WRIGHT, GJ HAUSMAN. Preadipocyte recruitment in stromal vascular cultures after depletion of committed preadipocytes by immunocytotoxicity. Obes Res. 1997;5:9-15. Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complementmediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 f 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 f 5.1/ unit area) after removal of preadipocytes. Immunocytochemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 f 45/unit area) than FBS alone (10.5 f 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentia-