Berberine (BBR), an isoquinoline alkaloid, has a wide range of pharmacological effects, yet its exact mechanism is unknown. In order to understand the anti-adipogenic effect of BBR, we studied the change of expression of several adipogenic enzymes of 3T3-L1 cells by BBR treatment. First, we measured the change of leptin and glycerol in the medium of 3T3-L1 cells treated with 1 micrometer, 5 micrometer and 10 micrometer concentrations of BBR. We also measured the changes of adipogenic and lipolytic factors of 3T3-L1. In 3T3-L1 cells, both leptin and adipogenic factors (SREBP-1c, C/EBP-alpha, PPAR-gamma, fatty acid synthase, acetyl-CoA carboxylase, acyl-CoA synthase and lipoprotein lipase) were reduced by BBR treatment. Glycerol secretion was increased, whereas expression of lipolytic enzymes (hormone-sensitive lipase and perilipin) mRNA was slightly decreased. Next, we measured the change of inflammation markers of 3T3-L1 cells by BBR treatment. This resulted in the down-regulation of mRNA level of inflammation markers such as TNF-alpha, IL-6, C- reactive protein and haptoglobin. Taken together, our data shows that BBR has both anti-adipogenic and anti-inflammatory effects on 3T3-L1 adipocytes, and the anti-adipogenic effect seems to be due to the down-regulation of adipogenic enzymes and transcription factors.
RNA, Messenger/genetics/metabolism ; Mice ; Leptin/secretion ; Inflammation Mediators/*metabolism ; Inflammation/genetics ; Glycerol/metabolism ; Gene Expression Regulation/*drug effects ; Cytokines/genetics ; Berberine/*pharmacology ; Animals ; Adipogenesis/drug effects/genetics ; Adipocytes/*drug effects/enzymology/*metabolism/secretion ; 3T3-L1 Cells

BACKGROUNDSteroids inhibit osteogenic differentiation and decrease bone formation while concomitantly inducing adipose deposition in osteocytes. This leads to the fatty degeneration and necrosis of bone cells commonly seen in osteonecrosis of the femoral head. The peroxisome proliferator-activated receptor-γ (PPARγ) is an adipogenic transcription factor linked to the development of this disease and responsible for inducing adipogenesis over osteogenesis in bone marrow mesenchymal stem cells (BMSCs). The aim of this study was to assess whether adipogenic differentiation could be suppressed, and thus osteogenic potential retained, by inhibiting PPARγ expression in BMSCs.

METHODSCells from the bone marrow of New Zealand rabbits were treated with 10(-7) mol/L dexamethasone and infected with one of three small interference RNA (siRNA) adenovirus vectors (S1, S2, and S3) or non-targeting control siRNA (Con) and compared with dexamethasone-treated (model) and untreated (normal) cells. Cells were grown for 21 days and stained with Sudan III for adipocyte formation. At various time points, cells were also assessed for changes in PPARγ, osteocalcin (OC), Runx2, alkaline phosphatase (ALP) activity, and triglyceride (TG) content.

RESULTSDexamethasone-treated model and control groups showed a significant increase in fatty acid-positive staining, which was inhibited in cells treated with PPARγ siRNA-treated, similar to normal untreated cells. All three siRNA groups significantly inhibited PPARγ mRNA and protein, adipocyte number, and TG content compared with the dexamethasone-treated model and control groups, matching that seen in normal cells. OC and Runx2 mRNA and protein, as well as ALP activity, were significantly higher in cells treated with siRNA against PPARγ, similar to that seen in the normal cells. These osteogenic markers were significantly lower in the dexamethasone-treated cell cultures.

CONCLUSIONSThe siRNA adenovirus vector targeting PPARγ can efficiently inhibit steroid-induced adipogenic differentiation in rabbit BMSCs and retain their osteogenic differentiation potential.

Adenoviridae ; genetics ; Adipogenesis ; drug effects ; genetics ; Animals ; Cell Differentiation ; drug effects ; genetics ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; PPAR gamma ; genetics ; metabolism ; pharmacology ; RNA, Small Interfering ; Rabbits ; Steroids

BACKGROUNDIn steroid-induced osteonecrosis, hypertrophy and hyperplasia of marrow fat cells and lipid deposition of osteocytes can be found in the femoral head. However, the precise reason is not clear yet. The aim of this study was to observe the effect of dexamethasone (Dex) on differentiation of marrow stromal cells (MSCs), and to investigate the pathobiological mechanism of steroid-induced osteonecrosis.

METHODSMSCs in cultures were treated with increasing concentrations of Dex (0, 10(-9), 10(-8), 10(-7), and 10(-6) mol/L) continuously for 21 days. The cells, which were exposed to 0 mol/L (control) or 10(-7) mol/L Dex for 4 - 21 days, were then cultured for 21 days without Dex. MSCs were stained with Sudan III. Number of adipocytes was counted under a light microscope. The activity of alkaline phosphatase (ALP) of MSCs treated with 0, 10(-8), 10(-7), and 10(-6) mol/L Dex for 12 days, and that treated with 0 mol/L and 10(-7) mol/L Dex for 8, 10, or 12 days were determined. The levels of triglycerides, osteocalcin and cell proliferation of MSCs treated with 0 mol/L and 10(-7) mol/L Dex were detected. The mRNA expression levels of adipose-specific 422 (aP2) gene and osteogenic gene type I collagen in MSCs treated with 0 mol/L and 10(-7) mol/L Dex for 6 days were analyzed by whole-cell dot-blot hybridization. Statistical analysis was performed using Student's t test and analysis of variance. P values less than 0.05 were considered significant statistically.

RESULTSThe number of adipocytes in cultures increased with the duration of MSCs' exposure to Dex and the concentration of Dex. The level of ALP activity in the MSCs decreased with concentration of Dex. In the control group, it was 8.69 times of that in the 10(-7) mol/L Dex group on day 12 (t = 20.51, P < 0.001). The level of triglycerides in 10(-7) mol/L Dex group was 3.40 times of that in the control (t = 11.00, P < 0.001). The levels of cell proliferation and osteocalcin in the control were 1.54 and 2.42 times of that in the 10(-7) mol/L Dex group respectively. As compared to the control, the mRNA expression of adipose-specific 422 (aP2) gene in 10(-7) mol/L Dex group was significantly increased (t = 36.48, P < 0.001), and that of osteogenic gene type I collagen was decreased (t = 42.07, P < 0.001).

CONCLUSIONSDex can directly induce the differentiation of MSCs into a large number of adipocytes and inhibit their osteogenic differentiation, which provide a novel explanation for the pathologic changes of steroid-induced osteonecrosis.

Adipogenesis ; drug effects ; Alkaline Phosphatase ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dexamethasone ; toxicity ; Female ; Mice ; Osteocalcin ; genetics ; Osteonecrosis ; chemically induced ; RNA, Messenger ; analysis ; Radioimmunoassay ; Stromal Cells ; cytology

PURPOSE: Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs). MATERIALS AND METHODS: Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates. RESULTS: ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)gamma expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARgamma, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARgamma agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types. CONCLUSION: The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.
Adipogenesis/*drug effects/genetics ; Adult ; Anilides/pharmacology ; Bone Marrow Cells/*cytology/drug effects/metabolism ; Butadienes/pharmacology ; Cell Differentiation/drug effects ; Cells, Cultured ; Chromans/pharmacology ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Female ; Humans ; Male ; Middle Aged ; Nitriles/pharmacology ; Osteogenesis/*drug effects/genetics ; PPAR gamma/agonists/antagonists & inhibitors ; Phosphorylation/drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells/*cytology/drug effects/*metabolism ; Thiazolidinediones/pharmacology

PURPOSE: Imbalances between osteogenic and adipogenic differentiation leads to diseases such as osteoporosis. The aim of our study was to demonstrate the differences in extracellular signal-regulated kinase (ERK) phosphorylation during both adipogenesis and osteogenesis of human bone marrow-derived stem cells (BMSCs). MATERIALS AND METHODS: Using troglitazone, GW9662 and U0126, we investigated their role in hBMSC differentiation to adipogenic and osteogenic fates. RESULTS: ERK1/2 inhibition by U0126 suppressed proliferator-activated receptor (PPAR)gamma expression and lipid accumulation, while it decreased the mRNA expression of adipogenic genes (lipoprotein lipase, PPARgamma, and adipocyte protein) and osteogenic genes (type I collagen and osteopontin). ERK phosphorylation was transient and decreased during adipogenesis, whereas it occurred steadily during osteogenesis. Troglitazone, a PPARgamma agonist, induced adipogenesis by inhibiting ERK phosphorylation even in an osteogenic medium, suggesting that ERK signaling needs to be shut off in order to proceed with adipose cell commitment. Cell proliferation was greatly increased in osteogenesis but was not changed during adipogenesis, indicating that ERK might play different roles in cellular proliferation and differentiation between the two committed cell types. CONCLUSION: The duration and magnitude of ERK activation might be a crucial factor for the balance between adipogenesis and osteogenesis in human bone marrow-derived stem cells.
Adipogenesis/*drug effects/genetics ; Adult ; Anilides/pharmacology ; Bone Marrow Cells/*cytology/drug effects/metabolism ; Butadienes/pharmacology ; Cell Differentiation/drug effects ; Cells, Cultured ; Chromans/pharmacology ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Female ; Humans ; Male ; Middle Aged ; Nitriles/pharmacology ; Osteogenesis/*drug effects/genetics ; PPAR gamma/agonists/antagonists & inhibitors ; Phosphorylation/drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells/*cytology/drug effects/*metabolism ; Thiazolidinediones/pharmacology

Lysimachia foenum-graecum has been used as an oriental medicine with anti-inflammatory effect. The anti-obesity effect of L. foenum-graecum extract (LFE) was first discovered in our screening of natural product extract library against adipogenesis. To characterize its anti-obesity effects and to evaluate its potential as an anti-obesity drug, we performed various obesity-related experiments in vitro and in vivo. In adipogenesis assay, LFE blocked the differentiation of 3T3-L1 preadipocyte in a dose-dependent manner with an IC50 of 2.5 microg/ml. In addition, LFE suppressed the expression of lipogenic genes, while increasing the expression of lipolytic genes in vitro at 10 microg/ml and in vivo at 100 mg/kg/day. The anti-adipogenic and anti-lipogenic effect of LFE seems to be mediated by the inhibition of PPARgamma and C/EBPalpha expression as shown in in vitro and in vivo, and the suppression of PPARgamma activity in vitro. Moreover, LFE stimulated fatty acid oxidation in an AMPK-dependent manner. In high-fat diet (HFD)-induced obese mice (n = 8/group), oral administration of LFE at 30, 100, and 300 mg/kg/day decreased total body weight gain significantly in all doses tested. No difference in food intake was observed between vehicle- and LFE-treated HFD mice. The weight of white adipose tissues including abdominal subcutaneous, epididymal, and perirenal adipose tissue was reduced markedly in LFE-treated HFD mice in a dose-dependent manner. Treatment of LFE also greatly improved serum levels of obesity-related biomarkers such as glucose, triglycerides, and adipocytokines leptin, adiponectin, and resistin. All together, these results showed anti-obesity effects of LFE on adipogenesis and lipid metabolism in vitro and in vivo and raised a possibility of developing LFE as anti-obesity therapeutics.
3T3-L1 Cells ; Adipogenesis/*drug effects ; Adipose Tissue/drug effects/metabolism ; Adipose Tissue, White ; Animals ; Anti-Obesity Agents/administration & dosage/pharmacology/*therapeutic use ; Body Weight/drug effects ; CCAAT-Enhancer-Binding Protein-alpha/genetics ; Cell Differentiation/drug effects ; Eating/drug effects ; Fatty Acids/metabolism ; Gene Expression/drug effects ; Lipid Metabolism/*drug effects ; Lipids ; Lipogenesis/drug effects ; Mice ; Mice, Inbred C57BL ; Obesity/prevention & control ; PPAR gamma/antagonists & inhibitors/genetics ; Plant Extracts/*pharmacology ; Plants, Medicinal ; Primulaceae/*chemistry

Parathyroid hormone-related protein (PTHrP) is synthesized by diverse tissues, and its processing produces several fragments, each with apparently distinct autocrine and paracrine bioactivities. In bone, PTHrP appears to modulate bone formation in part through promoting osteoblast differentiation. The putative effect of PTH-like and PTH-unrelated fragments of PTHrP on human mesenchymal stem cell (MSCs) is not well known. Human MSCs were treated with PTHrP (1-36) or PTHrP (107-139) or both (each at 10 nM) in osteogenic or adipogenic medium, from the start or after 6 days of exposure to the corresponding medium, and the expression of several osteoblastogenic and adipogenic markers was analyzed. PTHrP (1-36) inhibited adipogenesis in MSCs and favoured the expression of osteogenic early markers. The opposite was observed with treatment of MSCs with PTHrP (107-139). Moreover, inhibition of the adipogenic differentiation by PTHrP (1-36) prevailed in the presence of PTHrP (107-139). The PTH/PTHrP type 1 receptor (PTH1R) gene expression was maximum in the earlier and later stages of osteogenesis and adipogenesis, respectively. While PTHrP (107-139) did not modify the PTH1R overexpression during adipogenesis, PTHrP (1-36) did inhibit it; an effect which was partially affected by PTHrP (7-34), a PTH1R antagonist, at 1 microM. These findings demonstrate that both PTHrP domains can exert varying effects on human MSCs differentiation. PTHrP (107-139) showed a tendency to favor adipogenesis, while PTHrP (1-36) induced a mild osteogenic effect in these cells, and inhibited their adipocytic commitment. This further supports the potential anabolic action of the latter peptide in humans.
Adipogenesis/drug effects ; Alkaline Phosphatase/biosynthesis/genetics ; Antigens, Differentiation/biosynthesis/genetics ; Bone Marrow/pathology ; Cell Differentiation/drug effects ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/biosynthesis/genetics ; Culture Media ; Gene Expression Regulation ; Humans ; Lipoprotein Lipase/biosynthesis/genetics ; Mesenchymal Stem Cells/*drug effects/metabolism/pathology ; Osteoblasts/drug effects/*metabolism/pathology ; Osteogenesis/drug effects ; PPAR gamma/biosynthesis/genetics ; Parathyroid Hormone/*pharmacology ; Peptide Fragments/*pharmacology ; Receptor, Parathyroid Hormone, Type 1/antagonists & inhibitors

Obesity is associated with a number of metabolic abnormalities such as type 2 diabetes and has become a major health problem worldwide. In the present study, we investigated the effects of Epimedium koreanum Nakai (Herba Epimedii, HE) and its main constituent icariin on the adipocyte differentiation in 3T3-L1 preadipocytes. HE extract and icariin significantly reduced lipid accumulation and suppressed the expressions of PPARγ, C/EBPα, and SREBP-1c in 3T3-L1 adipocytes. They also inhibited fatty acid synthase (FAS), acyl-Co A synthase (ACS1), and perilipin. Moreover, HE extract and icariin markedly increased the phosphorylation of AMPK. These results indicated that HE extract and icariin can inhibit the adipocyte differentiation through downregulation of the adipogenic transcription factors, suggesting that HE containing icariin may be used as a potential therapeutic agent in the treatment and prevention of obesity.
3T3-L1 Cells ; Adipocytes ; cytology ; drug effects ; metabolism ; Adipogenesis ; drug effects ; Animals ; CCAAT-Enhancer-Binding Protein-alpha ; genetics ; metabolism ; Epimedium ; chemistry ; Flavonoids ; pharmacology ; Lipid Metabolism ; drug effects ; Mice ; PPAR gamma ; genetics ; metabolism ; Plant Extracts ; pharmacology ; Sterol Regulatory Element Binding Protein 1 ; genetics ; metabolism

PURPOSE: Osteoprotegerin (OPG), a potent inhibitor of osteoclastic bone resorption, has a variety of biological functions that include anti-inflammatory effects. Adipocytes and osteoblasts share a common origin, and the formation of new blood vessels often precedes adipogenesis in developing adipose tissue microvasculature. We examined whether OPG is secreted from adipocytes, therefore contributing to the prevention of neovascularization and protecting the vessels from intimal inflammation and medial calcification. MATERIALS AND METHODS: The mRNA expression of OPG and receptor activator of NF-kappaB ligand (RANKL) was measured in differentiated 3T3L1 adipocytes and adipose tissues. RESULTS: OPG mRNA expression increased with the differentiation of 3T3L1 adipocytes, while RANKL expression was not significantly altered. OPG mRNA was expressed at higher levels in white adipose tissue than in brown adipose tissue and was most abundant in the epididymal portion. In differentiated 3T3L1 adipocytes, Rosiglitazone and insulin reduced the OPG/RANKL expression ratio in a dose- and time- dependent manner. In contrast, tumor necrosis factor-alpha (TNF-alpha) increased the expression of both OPG and RANKL in a time-dependent manner. The OPG/RANKL ratio was at a maximum two hours after TNF-alpha treatment and then returned to control levels. Furthermore, OPG was abundantly secreted into the media after transfection of OPG cDNA with Phi C31 integrase into 3T3L1 cells. CONCLUSION: Our results indicate that OPG mRNA is expressed and regulated in the adipose tissue. Considering the role of OPG in obesity-associated inflammatory changes in adipose tissue and vessels, we speculate that OPG may have both a protective function against inflammation and anti-angiogenic effects on adipose tissue.
3T3-L1 Cells ; Adipocytes/cytology/drug effects/metabolism ; Adipogenesis/genetics ; Adipose Tissue/cytology/*metabolism ; Animals ; Cell Differentiation ; *Gene Expression Regulation/drug effects ; Hypoglycemic Agents/pharmacology ; Insulin/pharmacology ; Male ; Mice ; Osteoprotegerin/genetics/*metabolism ; RANK Ligand/metabolism ; Rats ; Rats, Sprague-Dawley ; Thiazolidinediones/pharmacology ; Transfection ; Tumor Necrosis Factor-alpha/pharmacology

Higher levels of body fat are associated with an increased risk for development numerous adverse health conditions. FTY720 is an immune modulator and a synthetic analogue of sphingosine 1-phosphate (S1P), activated S1P receptors and is effective in experimental models of transplantation and autoimmunity. Whereas immune modulation by FTY720 has been extensively studied, other actions of FTY720 are not well understood. Here we describe a novel role of FTY720 in the prevention of obesity, involving the regulation of adipogenesis and lipolysis in vivo and in vitro. Male C57B/6J mice were fed a standard diet or a high fat diet (HFD) without or with FTY720 (0.04 mg/kg, twice a week) for 6 weeks. The HFD induced an accumulation of large adipocytes, down-regulation of phosphorylated AMP-activated protein kinase alpha (p-AMPKalpha) and Akt (p-Akt); down-regulation of hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL) and perilipin mRNA as well as up-regulation of phosphorylated HSL (p-HSL, Ser563) and glycogen synthase kinase 3 alpha/beta (p-GSK3alpha/beta). All these effects were blunted by FTY720 treatment, which inhibited adipogenesis and promoted lipolysis. Also, FTY720 significantly decreased lipid accumulation in maturing preadipocytes. FTY720 down-regulated the transcriptional levels of the PPARgamma, C/EBPalpha and adiponectin, which are markers of adipogenic differentiation. FTY720 significantly increased the release of glycerol and the expression of the HSL, ATGL and perilipin, which are regulators of lipolysis. These results show that FTY720 prevented obesity by modulating adipogenesis and lipolysis, and suggest that FTY720 is used for the treatment of obesity.
3T3-L1 Cells ; AMP-Activated Protein Kinases/metabolism ; Adipocytes/*drug effects/physiology ; Adipogenesis/drug effects ; Animals ; Anti-Obesity Agents/*pharmacology/therapeutic use ; Antigens, Differentiation/genetics/metabolism ; Carrier Proteins/genetics/metabolism ; Cell Size ; Diet, High-Fat/adverse effects ; Disease Models, Animal ; Enzyme Activation ; Gene Expression Regulation, Enzymologic/drug effects ; Glycogen Synthase Kinase 3/genetics/metabolism ; Lipase/genetics/metabolism ; Lipolysis/drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Obesity/etiology/metabolism/*prevention & control ; Phosphoproteins/genetics/metabolism ; Phosphorylation ; Propylene Glycols/*pharmacology/therapeutic use ; Protein Processing, Post-Translational ; Proto-Oncogene Proteins c-akt/metabolism ; Sphingosine/*analogs & derivatives/pharmacology/therapeutic use ; Sterol Esterase/metabolism