OBJECTIVE: We aimed to explore how fermented barley extracts with Lactobacillus plantarum dy-1 (LFBE) affected the browning in adipocytes and obese rats. METHODS: In vitro, 3T3-L1 cells were induced by LFBE, raw barley extraction (RBE) and polyphenol compounds (PC) from LFBE to evaluate the adipocyte differentiation. In vivo, obese SD rats induced by high fat diet (HFD) were randomly divided into three groups treated with oral gavage: (a) normal control diet with distilled water, (b) HFD with distilled water, (c) HFD with 800 mg LFBE/kg body weight (bw). RESULTS: In vitro, LFBE and the PC in the extraction significantly inhibited adipogenesis and potentiated browning of 3T3-L1 preadipocytes, rather than RBE. In vivo, we observed remarkable decreases in the body weight, serum lipid levels, white adipose tissue (WAT) weights and cell sizes of brown adipose tissues (BAT) in the LFBE group after 10 weeks. LFBE group could gain more mass of interscapular BAT (IBAT) and promote the dehydrogenase activity in the mitochondria. And LFBE may potentiate process of the IBAT thermogenesis and epididymis adipose tissue (EAT) browning via activating the uncoupling protein 1 (UCP1)-dependent mechanism to suppress the obesity. CONCLUSION These results demonstrated that LFBE decreased obesity partly by increasing the BAT mass and the energy expenditure by activating BAT thermogenesis and WAT browning in a UCP1-dependent mechanism.
3T3 Cells ; Adipocytes ; drug effects ; physiology ; Adipose Tissue, Brown ; drug effects ; physiology ; Adipose Tissue, White ; drug effects ; physiology ; Animal Feed ; analysis ; Animals ; Anti-Obesity Agents ; administration & dosage ; metabolism ; Cell Differentiation ; drug effects ; Diet ; Fermentation ; Hordeum ; chemistry ; Lactobacillus plantarum ; chemistry ; Male ; Mice ; Obesity ; drug therapy ; genetics ; Plant Extracts ; chemistry ; Probiotics ; administration & dosage ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Uncoupling Protein 1 ; genetics ; metabolism

OBJECTIVETo investigate the potential role of nucleotide-binding oligomerization domain 1 (NOD1), a component of the innate immune system, in mediating lipid-induced insulin resistance in adipocytes.

METHODSAdipocytes from Toll-like receptor 4 deficiency mice were used for stimulation experiments. The effect of oleate/palmitate mixture on nuclear factor-κB (NF-κB) activation was analyzed by reporter plasmid assay. The release of proinflammatory chemokine/cytokines production was determined by using real-time PCR. Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[3H] glucose uptake assay. Chemokine/cytokine expression and glucose uptake in adipocytes transfected with small interfering RNA (siRNA) targeting NOD1 upon fatty acids treatment were analyzed.

RESULTSOleate/palmitate mixture activated the NF-κB pathway and induced interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 mRNA expressions in adipocytes from mice deficient in Toll-like receptor 4, and these effects were blocked by siRNA targeting NOD1. Furthermore, saturated fatty acids decreased the ability of insulin-stimulated glucose uptake. Importantly, siRNA targeting NOD1 partially reversed saturated fatty acid-induced suppression of insulin-induced glucose uptake.

CONCLUSIONNOD1 might play an important role in saturated fatty acid-induced insulin resistance in adipocytes, suggesting a mechanism by which reduced NOD1 activity confers beneficial effects on insulin action.

Adipocytes ; drug effects ; metabolism ; Animals ; Fatty Acids ; pharmacology ; Insulin Resistance ; Male ; Mice ; Mice, Inbred C57BL ; NF-kappa B ; physiology ; Nod1 Signaling Adaptor Protein ; physiology ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; physiology

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

OBJECTIVETo investigate the effect of adipose stromal vascular fraction cells (SVFs) with VEGF on the neovascularization of free fat transplantation.

METHODSSVFs were obtained from subcutaneous fat and labelled with DiI. 0.3 ml autologous fat tissue was mixed with 0.2 ml cells: 1) autologous SVFs with VEGF (Group A); 2) autologous SVFs (Group B); 3) complete DMEM (Group C) And then the mixture was injected randomly under the back skin of 12 nude mice. The transplanted fat tissue in three groups was harvested at 2 months after implantation. Wet weight and diameter of fat grafts was measured. After HE and CD31 staining,blood vessel density, viable adipocytes and fibrous proliferation were observed.

RESULTSTrace of SVFs labeled by DiI in vivo could be detected by fluorescent microscope. The wet weight of fat grafts was (191.90 +/- 9.81) mg in group A, (177.01 +/- 10.50) mg in group B, and (92.05 +/- 8.30) mg in group C (P<0.01). The diameter of fat grafts was (0.49 +/- 0.24) cm in group A, (0.40 +/- 0.26) cm in group B, and (0.32 +/- 0.28) cm in group C (P<0.01). Histological analysis showed the blood vessel density was (14.58 +/- 2.06)/HPL in group A, (11.55 +/- 2.18)/HPL in group B, (7.87 +/- 1.55)/HPL in group C. Compared with group B and group C, group A had more adipose tissue with less fat necrosis and fibrosis and had significantly higher capillary density.

CONCLUSIONSThe autologous adipose stromal vascular fraction cells with VEGF could improve the neovascularization of free fat significantly. It indicates a wide clinical application in the future.

Adipocytes ; Adipose Tissue ; anatomy & histology ; blood supply ; transplantation ; Animals ; Capillaries ; Graft Survival ; Mice ; Mice, Nude ; Neovascularization, Physiologic ; drug effects ; physiology ; Organ Size ; Stromal Cells ; transplantation ; Vascular Endothelial Growth Factor A ; therapeutic use

Leptin is an adipocytokine that regulates body weight, and maintains energy homeostasis by promoting reduced food intake and increasing energy expenditure. Leptin expression and secretion is regulated by various factors including hormones and fatty acids. Butyrate is a short-chain fatty acid that acts as source of energy in humans. We determined whether this fatty acid can play a role in leptin expression in fully differentiated human adipocytes. Mature differentiated adipocytes were incubated with or without increasing concentrations of butyrate. RNA was extracted and leptin mRNA expression was examined by Northern blot analysis. Moreover, the cells were incubated with regulators that may affect signals which may alter leptin expression and analyzed with Northern blotting. Butyrate stimulated leptin expression, and stimulated mitogen activated protein kinase (MAPK) and phospho-CREB signaling in a time-dependent manner. Prior treatment of the cells with signal transduction inhibitors as pertusis toxin, Gi protein antagonist, PD98059 (a MAPK inhibitor), and wortmannin (a PI3K inhibitor) abolished leptin mRNA expression. These results suggest that butyrate can regulate leptin expression in humans at the transcriptional level. This is accomplished by: 1) Gi protein-coupled receptors specific for short-chain fatty acids, and 2) MAPK and phosphatidylinositol-3-kinase (PI3K) signaling pathways.
Adipocytes/*metabolism ; Azo Compounds ; Butyric Acid/*pharmacology ; CREB-Binding Protein/genetics/metabolism ; Cell Differentiation ; Cells, Cultured ; Gene Expression Regulation/*drug effects/physiology ; Humans ; Leptin/genetics/*metabolism ; Mitogen-Activated Protein Kinase Kinases/genetics/metabolism ; Phosphatidylinositol 3-Kinases/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Signal Transduction/*physiology ; Staining and Labeling

We stimulated preadipocyte of mice with calcium acetate, p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580, the paralysors and excitomotors of calcium channel. Then we detected expression level of preadipocyte differentiation's marker genes and calcium signal related acceptor genes by real-time PCR, and determined intracellular free Ca2+ concentration ([Ca2+]i]) with Fura-2/AM, intracellular lipid accumulation by oil red O staining. Our aim was to investigate the potential mechanism between calcium signal and preadipocyte differentiation. The results indicated that the paralysors and excitomotors of calcium channel changed the expression level of lipoprotein lipase (LPL), peroxisome proliferators-activated receptor gamma (PPARgamma), fatty acid synthetase (FAS), and the lipid accumulation, markedly. Compared with exocellular Ca2+'s decrease, inhibited intracellular Ca2+'s liberation can promoted preadipocyte differentiation (P < 0.01), and compared with intracellular Ca2+'s increase, promoted exocellular Ca2+'s ingest inhibited preadipocyte differentiation (P < 0.01). SB203580 degraded [Ca2+]i, promoted differentiation marker genes' expression and lipid accumulation in preadipocyte (P < 0.01). But calcium signal didn't have effects to vitamin D receptor (VDR) and extracellular Ca2+-sensing receptor (CaSR)'s expression. It indicated that calcium signal may effect preadipocyte different and lipid accumulation by p38 MAPK pathway.
Adipocytes ; cytology ; Animals ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; Cell Differentiation ; physiology ; Cells, Cultured ; Imidazoles ; pharmacology ; Lipids ; biosynthesis ; Mice ; Pyridines ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

OBJECTIVETo study the effect of a new obesity-related gene NYGGF4 on the insulin sensitivity and secretory function of adipocytes.

METHODS3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1; control group) or an NYGGF4 expression vector (NYGGF4-pcDNA3.1) were cultured in vitro and differentiated into the matured adipocytes with the standard insulin plus dexamethasone plus 3-isobutyl-methylxanthine (MDI) induction cocktail. 2-deoxy-D-[3H] glucose uptake was determined by liquid scintillation counting. Western blot was performed to detect the protein content and translocation of glucose transporter 4 (GLUT4). The supernatant concentrations of TNF-alpha, IL-6, adiponectin and resistin were measured using ELISA.

RESULTSNYGGF4 over-expression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. NYGGF4 over-expression impaired insulin-stimulated GLUT4 translocation without affecting the total protein content of GLUT4. The concentrations of TNF-alpha, IL-6, adiponectin and resistin in the culture medium of 3T3-L1 transfected with NYGGF4 were not significantly different from those in the control group.

CONCLUSIONSNYGGF4 over-expression impairs the insulin sensitivity of 3T3-L1 adipocytes through decreasing GLUT4 translocation and had no effects on the secretory function of adipocytes.

3T3-L1 Cells ; Adipocytes ; drug effects ; secretion ; Adiponectin ; secretion ; Animals ; Carrier Proteins ; genetics ; physiology ; Glucose ; metabolism ; Glucose Transporter Type 4 ; analysis ; metabolism ; Insulin ; pharmacology ; Interleukin-6 ; secretion ; Mice ; Resistin ; analysis ; Transfection ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo optimize the condition for inducing the differentiation of 3T3-L1 preadipocytes into adipocytes and study the expression of PTEN tumor suppression gene in this process, aiming to understand the regulatory role of PTEN in normal adipocyte differentiation and collect laboratory evidence for developing drugs targeting PTEN.

METHODSThe differentiation of 3T3-L1 preadipocytes cultured in high-glucose DMEM were induced according to 2 protocols with different combinations of dexamethasone, isobutylmethylxanthine (IBMX) and insulin, and the resultant adipocytes were identified by oil red O staining. The total proteins of 3T3-L1 were extracted and analyzed by Western blotting, and PTEN homology between mice and human was analyzed by bioinformatic method.

RESULTSFor optimized 3T3-L1 differentiation, 3T3-L1 cells were initially induced with the combination of 1 micromol/L dexamethasone, 0.5 mmol/L IBMX and 5 microg/ml insulin for 48 h, followed by treatment with 5 microg/ml insulin in 4.5 g/L glucose DMEM for 48 h, which resulted in high differentiation rate of 3T3-L1 cells (up to 90% on the 10th day) with unified morphology and size. PTEN expression varied quantitatively in the process of differentiation, especially low on the 12th day as compared with those measured on days 4, 6 and 9. The mice PTEN mRNA shared 96% homology and PTEN amino acid 100% homology with their human counterparts.

CONCLUSIONEndogenous PTEN expression is down-regulated during 3T3-L1 differentiation, suggesting that PTEN may enhance insulin sensitivity and promote adipogenesis under physiological conditions.

3T3-L1 Cells ; Adipocytes ; cytology ; drug effects ; metabolism ; Animals ; Blotting, Western ; Cell Differentiation ; drug effects ; genetics ; physiology ; Glucose ; pharmacology ; Humans ; Mice ; PTEN Phosphohydrolase ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics

In adipocytes, insulin stimulates glucose transport primarily by promoting the translocation of GLUT4 to the plasma membrane. Requirements for Ca2+/ calmodulin during insulin-stimulated GLUT4 translocation have been demonstrated; however, the mechanism of action of Ca2+ in this process is unknown. Recently, myosin II, whose function in non-muscle cells is primarily regulated by phosphorylation of its regulatory light chain by the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), was implicated in insulin-stimulated GLUT4 translocation. The present studies in 3T3- F442A adipocytes demonstrate the novel finding that insulin significantly increases phosphorylation of the myosin II RLC in a Ca2+-dependent manner. In addition, ML-7, a selective inhibitor of MLCK, as well as inhibitors of myosin II, such as blebbistatin and 2,3-butanedione monoxime, block insulin- stimulated GLUT4 translocation and subsequent glucose transport. Our studies suggest that MLCK may be a regulatory target of Ca2+/calmodulin and may play an important role in insulin-stimulated glucose transport in adipocytes.
Protein Transport/drug effects ; Phosphorylation ; Naphthalenes/pharmacology ; Myosin-Light-Chain Kinase/antagonists & inhibitors/*metabolism ; Myosin Type II/*metabolism ; Mice ; Insulin/*pharmacology ; Glucose Transporter Type 4/*metabolism ; Enzyme Inhibitors/pharmacology ; Dose-Response Relationship, Drug ; Calmodulin/antagonists & inhibitors/physiology ; Azepines/pharmacology ; Animals ; Adipocytes/cytology/*drug effects/metabolism ; 3T3 Cells

People with upper body or visceral obesity have a much higher risk of morbidity and mortality from obesity-related metabolic disorders than those with lower body obesity. In an attempt to develop therapeutic strategies targeting visceral obesity, depot- specific differences in the expression of genes in omental and subcutaneous adipose tissues were investigated by DNA array technology, and their roles in adipocyte differentiation were further examined. We found that levels of metallothionein-II (MT-II) mRNA and protein expression were higher in omental than in subcutaneous adipose tissues. The study demonstrates that MT-II may play an important role in adipocyte differentiation of 3T3L1 preadipocytes, and that N-acetylcysteine (NAC) inhibits the adipocyte differentiation of 3T3L1 cells by repressing MT-II in a time- and dose-dependent manner. Furthermore, the intraperitoneal administration of NAC to rats and mice resulted in a reduction of body weights, and a marked reduction in visceral fat tissues. These results suggest that MT-II plays important roles in adipogenesis, and that NAC may be useful as an anti-obesity drug or supplement.
Viscera/drug effects/metabolism ; Time Factors ; Subcutaneous Fat/drug effects ; Rats, Sprague-Dawley ; Rats ; Middle Aged ; Mice, Inbred C57BL ; Mice ; Metallothionein/*genetics/metabolism/physiology ; Male ; Humans ; Female ; Down-Regulation/drug effects/genetics ; Dose-Response Relationship, Drug ; Cell Differentiation/drug effects ; Body Weight/drug effects ; Anti-Obesity Agents/*pharmacology ; Animals ; Aged ; Adipose Tissue/cytology/drug effects/metabolism ; Adipocytes/cytology/drug effects/metabolism ; Acetylcysteine/*pharmacology ; 3T3-L1 Cells