Adipocytes ; cytology ; Cell Line ; Endothelial Cells ; cytology ; drug effects ; Epithelial-Mesenchymal Transition ; drug effects ; Humans ; Melatonin ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism

OBJECTIVETo study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs).

METHODSADSCs were isolated from human abdominal adipose tissue and cultured. The immunophenotype and adipose induced-differentiation were identified, and the third generation cells were collected. The collected cells were assigned to 1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol/L insulin groups according to the concentration of added insulin. When cells grew into 70% confluence in conventional medium, ADSCs were cultured further in serum-free DMEM containing insulin in different concentrations for 3 days. ADSCs cultured in medium without insulin were used as control group. Secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) of ADSCs were determined by enzyme-linked immunosorbent assay. The effects of the supernatant fluid of ADSCs' nutrient solution on the proliferation and collagen synthesis of the cultured fibroblast were detected by MTT chromatometry and hydroxyproline chromatometry.

RESULTSThe secretion amounts of VEGF and HGF of ADSCs in 1 x 10(-8) and 1 x 10(-7) mol/L insulin groups [(471 +/- 41, 762 +/- 66 ng/L), (643 +/- 64, 930 +/- 67 ng/L), respectively] were significantly higher as compared with those in control group (286 +/- 47, 577 +/- 84 ng/L) (P < 0.05 or P < 0.01). No change occurred in the secretion amount of VEGF and HGF of ADSCs in 1 x l0(-6) mol/L insulin group (P > 0.05). The supernatant fluid of ADSCs' nutrient medium of 1 x 10(-8), 1 x 10(-7) mol/L insulin groups showed obvious stimulative effect on the proliferation and collagen synthesis of fibroblasts, and it was most obvious in the 1 x 10(-7) mol/L group (P < 0.05 or P < 0.01).

CONCLUSIONSInsulin in the concentrations of 1 x 10(-8) and 1 x 10(-7) mol/L can notably promote ADSCs' function of secreting VEGF and HGF.

Adipocytes ; cytology ; drug effects ; secretion ; Cells, Cultured ; Fibroblasts ; cytology ; Hepatocyte Growth Factor ; metabolism ; Humans ; Insulin ; pharmacology ; Stem Cells ; cytology ; drug effects ; secretion ; Vascular Endothelial Growth Factor A ; metabolism

To explore the effect of chronic high dose of insulin on lipolysis in porcine adipocytes and the underlying molecular regulation mechanisms, we cultured primary porcine adipocytes and incubated them with different concentrations of insulin (0, 200, 400, 800, 1600 nmol/L) for 24-96 h in the absence or presence of specific protein kinase A (PKA) inhibitor or extracellular signal-related kinase (ERK) inhibitor. Then, we measured the glycerol release into the culture media as an indicator of the lipolysis, and observed the lipid accumulation morphology by phase-contrast microscopy. Further, we analyzed the gene expressions of perilipin A and peroxisome proliferator-activated receptor-gamma 2 (PPAR gamma 2) with semi-quantitative RT-PCR and Western blotting, respectively. The results showed that chronic high dose of insulin stimulated lipolysis in differentiated porcine adipocytes in a dose- and time-dependent manner, and significantly attenuated the lipolytic response to isoprenaline. Meanwhile, the protein and mRNA expressions of PPAR gamma 2 and perilipin A were significantly reduced. In addition, both PKA and ERK inhibitors significantly suppressed insulin-stimulated lipolysis, however, only ERK inhibitor reversed the insulin-induced down-regulation of perilipin A. These findings imply that chronic high dose of insulin stimulates lipolysis in porcine adipocytes by repressing perilipin A, which is involved in ERK pathway.
Adipocytes ; cytology ; drug effects ; metabolism ; Animals ; Carrier Proteins ; Dose-Response Relationship, Drug ; Down-Regulation ; drug effects ; Insulin ; pharmacology ; Lipolysis ; drug effects ; Perilipin-1 ; Phosphoproteins ; metabolism ; Swine

We demonstrated the effect of resveratrol on porcine primary preadipocytes apoptosis, to study the intracellular molecular mechanism. Porcine primary preadipocyte was treated with different concentration of resveratrol (0 micromol/L, 50 micromol/L, 100 micromol/L, 200 micromol/L, 400 micromol/L). We used optical microscope and fluorescence microscope to observe morphological changes during apoptosis after Hoechst 33258 Fluorescent dyes staining; and RT-PCR and Western blotting to measure the expression of apoptosis-associated gene sirt1, caspase-3, bcl-2, bax, p53, NF-kappaB. Primary preadipocyte apoptosis was apparent, accompanied by reduced cell volume, chromatin condensation, and nuclear shrinkage. Compared to the control and low concentration group, high dose group (200 micromol/L) significantly increased the ratio of primary preadipocyte apoptosis. The expression of sirt1, caspase-3, and bax was up-regulated markedly in response to resveratrol; in contrast, apoptotic inhibitor bcl-2, p53, NF-kappaB down-regulated. We further proved fact that resveratrol can specifically promote the activity of sirt1; moreover, activated sirt1 modulates the activity of caspase-3 and bcl-2 family, involving in transcriptional regulation of p53 and NF-kappaB through antagonizing factor-induced acetylation. Taken together, our data established resveratrol as new regulator in porcine primary preadipocyte apoptosis via activating the expression of sirt1, modulating activity of apoptotic-associated factor.
Adipocytes ; cytology ; Adipogenesis ; Animals ; Antioxidants ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cells, Cultured ; Sirtuin 1 ; metabolism ; Stilbenes ; pharmacology ; Swine

AIMTo investigate the effect of interleukin-8 (IL-8) on the differentiation and clonal expansion of 3T3-L1 preadipocyte during the differentiation period.

METHODSThe morphological changes of 3T3-L1 cells during differentiation after the treatment of IL-8 was observed by Oil-Red O staining. Glycerol-3-phosphate dehydrogenase (GPDH) activity was measured by a spectrophotometric method. MTT method and 3H-TdR incorporation were applied to examine the changes of cell proliferation and DNA synthesis in clonal expansion of 3T3-L1 cells. Cell cycle analysis was taken by flow cytometry.

RESULTSIL-8 could inhibit the differentiation and GDPH activity in a dose dependent manner. IL-8 decreased the cell proliferation and DNA synthesis in clonal expansion after induction. Also, the proportion of cells in G1 phase was increased and that of cells in S and G2 phase was declined after the treatment of IL-8.

CONCLUSIONIL-8 inhibits the differentiation of 3T3-L1 preadipocytes by decreasing the clonal expansion of the cells.

3T3-L1 Cells ; Adipocytes ; cytology ; metabolism ; Animals ; Cell Cycle ; Cell Differentiation ; Cell Proliferation ; drug effects ; Interleukin-8 ; pharmacology ; Mice

OBJECTIVETo observe the impact of Rhizoma Coptidis (drug-chemical extract parts-components) on 3T3-L1 pre-adipocytes differentiation and adipocytes insulin resistance, and reveal the pharmacodyamic material basis of Rhizoma Coptidis on insulin resistance.

METHOD3T3-L1 pre-adipocytes were well cultured, and then induced to differentiate into fat cells by using dexamethasone, 3-isobutyl-1-methyl-xanthine and insulin together, and establish the insulin resistance model. Based on the experience of traditional medicine use, the adipocytes differentiation and the glucose consumption in the cell culture medium were observed independently.

RESULTAqueous extract, different chemical extract fraction and different alkaloid extract from the herb showed inhibitory effects on 3T3-L1 pre-adipocytes differentiation, especially the compound coptisine significantly inhibited the differentiation in the concentration of 16.5 micromol x L(-1), but non-alkaloid extract from the herb promoted cell differentiation significantly in the concentration of 6.0 micromol x L(-1). Each treatment group, especially jatrorrhizine hydrochloride (in the concentration of 10.5 micromol x L(-1)) significantly decreased the concentration of glucose in 3T3-L1 adipocytes culture, at the same time improved insulin resistance. These effects are similar to the role of rosiglitazone maleate.

CONCLUSIONRhizoma Coptidis significantly improved insulin resistance, prevented pre-adipocytes differentiation. Its efficacy may be the synergistic effect of various components. Meanwhile, its role in inhibiting differentiation of pre-adipocytes indicates that coptis to increasing glucose uptake dose not cause fat accumulation and weight increasing. This has some clinical significance in the insulin resistance and metabolic syndrome.

3T3-L1 Cells ; Adipocytes ; cytology ; drug effects ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Glucose ; metabolism ; Insulin Resistance ; Mice ; Rhizome ; chemistry

Leptin, a cytokine predominantly secreted from fat tissue, plays an important role in regulating organism energy balance. Leptin can stimulate lipolysis, but the mechanism is unclear. In order to study the molecular mechanism of leptin stimulating lipolysis, we systemically studied the mRNA expression of key lipolytic enzymes. Morphological observation, Oil Red O staining and RT-PCR were used to identify pig primary adipocytes; commercial kits were used to measure the glycerol and FFA release; Semiquantitative RT-PCR was used to detect the mRNA expression of key lipolytic enzymes. The results showed that 100 nmol/L leptin up-regulated the mRNA expression of ATGL, TGH-2, HSL, MGL and LPL (P<0.01), but down-regulated the Perilipin mRNA expression (P<0.01). At the same time, leptin promoted the glycerol release in a dose dependent manner (P<0.01), but had no effect on the FFA release (P>0.05). These indicate that leptin may mainly stimulate lipolysis in pig primary adipocytes by up-regulating the expression of ATGL, MGL, LPL and down-regulating the expression of Perilipin. The unchanged FFA release may be resulted from Leptin promoting UCPs mRNA expression and increasing FFA expenditure.
Adipocytes ; cytology ; enzymology ; metabolism ; Animals ; Animals, Newborn ; Cells, Cultured ; Leptin ; pharmacology ; Lipase ; genetics ; metabolism ; Lipolysis ; drug effects ; Male ; Monoacylglycerol Lipases ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Swine

The present study was designed to synthesize and evaluate a series of benzylisoquinoline derivatives. These compounds were synthesized by Bischler-Napieralski cyclization to yield 1-benzyl-3,4-dihydroisoquinolines, and the products were obtained by reductions. All these compounds were identified by MS, (1)H NMR and (13)C NMR. The inhibitory activities on pancreatic lipase and preadipocyte proliferation for the synthesized compounds and alkaloids from Nulembo nucifera were assessed in vitro. Most of the compounds showed inhibitory activities on both pancreatic lipase and preadipocyte proliferation. Particularly, compounds 7p-7u and 9d-9f exhibited significant inhibitory activity on pancreatic lipase while compounds 7c, 7d, 7f, 7g, 7i, and 7j potently inhibited the proliferation of 3T3-L1 preadipocytes. Our results provided a basis for future evaluation and development of these compounds as leads for therapeutics for human diseases.
Adipocytes ; cytology ; drug effects ; Benzylisoquinolines ; chemical synthesis ; chemistry ; pharmacology ; Cell Proliferation ; drug effects ; Enzyme Inhibitors ; chemical synthesis ; chemistry ; pharmacology ; Humans ; Lipase ; antagonists & inhibitors ; metabolism ; Structure-Activity Relationship

OBJECTIVETo study the effect and mechanism of berberine (BER) on the proliferation and differentiation of adipocytes.

METHODSThe proliferation of 3T3-L1 pre-adipocytes was detected by XTT method. Lipid droplets accumulated in the cytoplasm of adipocytes in the differentiating process were observed by oil red O staining and quantified by colorimetry. The expressions of peroxisome proliferation activated receptor gamma (PPARgamma), CAAT/enhancer binding protein alpha (C/EBPalpha) mRNA and protein were detected by Real-time PCR and Western blotting respectively.

RESULTSIntervention with BER in concentration below 10 micromol/L for 24 h showed insignificant effect on the proliferation of adipocytes, as compared with that in the control group (P > 0.05); but that in concentrations 20, 40 and 80 micromol/L revealed significant suppressive effect; that in different concentrations acting for 48 h and 72 h could affect the proliferation and the effect displayed a dose-dependent manner, i. e. the higher the concentration of BER, the more apparent the suppression, showing significant difference as compared with those in the control group (P <0.05 or P <0.01). The pre-adipocyte treated with 10 micromol/L BER showed that the lipid droplets in the cytoplasm significantly lessened, so did the expression of differentiation related factor PPAR gamma mRNA as well as the expressions of C/EBPalpha mRNA and protein, as compared with those in the blank control group and the group intervened with rosiglitazone, the difference was significant (P < 0.05 or P < 0.01).

CONCLUSIONSBER can suppress the proliferation and differentiation of 3T3-L1 pre-adipocytes, reduce the accumulation of lipid drops in the adipocyte differentiating process, which may be associated with its effects in decreasing the expressions of adipocyte differentiation related gene PPARgamma, C/EBPalpha mRNA and protein. The study provides a basis for applying BER on the prevention and treatment of such metabolic related diseases as obesity.

3T3-L1 Cells ; Adipocytes ; cytology ; drug effects ; metabolism ; Animals ; Berberine ; pharmacology ; CCAAT-Enhancer-Binding Protein-alpha ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Gene Expression ; drug effects ; Mice ; PPAR gamma ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo study the effects and mechanisms of Huanglian Jiedu Decoction (HJD) and its disassembled recipes containing serums on the proliferation and differentiation of preadipocytes.

METHODSHJD and its disassembled recipes containing serums were prepared. The 3T3-L1 preadipocytes were cultured. The proliferation of 3T3-L1 preadipocytes was detected by methyl thiazolyl tetrazolium (MTT) method. The accumulation of lipid droplets in the cytoplasm of differentiated preadipocytes was observed by oil red O staining and quantitatively analyzed by colorimetry. The mRNA expressions of peroxisome proliferation activated receptor y (PPAR gamma) and CAAT/enhancer binding protein (C/EBP alpha) were detected by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSIntervention with serum containing HJD, Phellodendron amurense Rupr plus Gardenia jasminoides Ellis, or Gardenia jasminoides Ellis showed significantly stimulative effects on the proliferation of preadipocytes, as compared with that in the blank control group (P<0.05, P<0.01). The preadipocytes treated with serum containing HJD, Phellodendron amurense Rupr plus Gardenia jasminoides Ellis, Coptis chinensis Franch or Gardenia jasminoides Ellis showed that the lipid droplets in the cytoplasm were significantly lessened, so did the mRNA expressions of PPAR gamma and C/EBP alpha when compared with the blank control group (P<0.05, P<0.01).

CONCLUSIONSHJD promoted the proliferation of preadipocytes, decreased the accumulation of lipid droplets during the differentiation of adipocytes, and inhibited the differentiation of adipocytes, which might be associated with its effects on decreasing the mRNA expressions of PPAR gamma and C/EBP alpha. Phellodendron amurense Rupr and Gardenia jasminoides Ellis were the main components of HJD playing these roles.

3T3-L1 Cells ; Adipocytes ; cytology ; drug effects ; metabolism ; Animals ; CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Mice ; PPAR gamma ; metabolism ; Rats ; Rats, Sprague-Dawley ; Serum