BACKGROUND/OBJECTIVE: This study was designed to investigate how a Portulaca oleracea L. extract (POE) stimulates insulin secretion in INS-1 pancreatic β-cells. MATERIALS/METHOD: INS-1 pancreatic β-cells were incubated in the presence of various glucose concentrations: 1.1 or 5.6, 16.7 mM glucose. The cells were treated with insulin secretagogues or insulin secretion inhibitor for insulin secretion assay using an insulin ELISA kit. In order to quantify intracellular influx of Ca2+ caused by POE treatment, the effect of POE on intracellular Ca2+ in INS-1 pancreatic β-cells was examined using Fluo-2 AM dye. RESULTS: POE at 10 to 200 µg/mL significantly increased insulin secretion dose-dependently as compared to the control. Experiments at three glucose concentrations (1.1, 5.6, and 16.7 mM) confirmed that POE significantly stimulated insulin secretion on its own as well as in a glucose-dependent manner. POE also exerted synergistic effects on insulin secretion with secretagogues, such as L-alanine, 3-isobutyl-1-methylxanthine, and especially tolbutamide, and at a depolarizing concentration of KCl. The insulin secretion caused by POE was significantly attenuated by treatment with diazoxide, an opener of the K+ ATP channel (blocking insulin secretion) and by verapamil (a Ca2+ channel blocker). The insulinotropic effect of POE was not observed under Ca2+-free conditions in INS-1 pancreatic β-cells. When the cells were preincubated with a Ca2+ fluorescent dye, Fluo-2 (acetoxymethyl ester), the cells treated with POE showed changes in fluorescence in red, green, and blue tones, indicating a significant increase in intracellular Ca2+, which closely correlated with increases in the levels of insulin secretion. CONCLUSIONS: These findings indicate that POE stimulates insulin secretion via a K+ ATP channel-dependent pathway in INS-1 pancreatic β-cells.
1-Methyl-3-isobutylxanthine ; Adenosine Triphosphate ; Alanine ; Calcium Channels ; Diabetes Mellitus ; Diazoxide ; Enzyme-Linked Immunosorbent Assay ; Fluorescence ; Glucose ; Insulin* ; Portulaca* ; Tolbutamide ; Verapamil

OBJECTIVETo investigate the optimal conditions for establishing insulin-resistant 3T3-L1 adipocytes.

METHOSDexamethason (DEX), 3-isobutyl-methylxanthine (IBMX) and different concentrations of insulin (10(-8), 10(-7), and 10(-6) mol·L(-1)) were used to induce 3T3-L1 preadipocytes into mature adipocytes identified by oil red O staining. We established insulin- resistant 3T3-L1 adipocytes cell model (IR-3T3-L1) by exposing the cells to 1µmol·L(-1) DEX, and the changes of glucose concen- tration in the cell culture were determined by glucose oxidase-peroxidase (GOD-POD) assay.

RESULTSTreatment of 3T3-L1 cells with DEX, IBMX and 10(-6) mol·L(-1)) insulin for 9 days resulted in the differentiation of >90% of the cells into mature adipocytes. IR-3T3-L1 cells cultured for 96 h in the culture media containing 1 µmol·L(-1) DEX showed significantly increased glucose consumption (P=0.0003) as compared with the control group at 36 h (P<0.001).

CONCLUSION3T3-L1 cells can be induced into mature adipocytes by exposure to 1 µmol·L(-1) DEX, 0.5 mmol·L(-1) IBMX and 10(-6) mol·L(-1)) insulin. A 96 h exposure to 1 µmol·L(-1) DEX can induce 3T3-L1 adipocytes to acquire insulin resistance that can be maintained for 36 h.

1-Methyl-3-isobutylxanthine ; chemistry ; 3T3-L1 Cells ; Adipocytes ; cytology ; Animals ; Cell Differentiation ; Culture Media ; chemistry ; Dexamethasone ; chemistry ; Glucose ; chemistry ; Insulin ; chemistry ; Insulin Resistance ; Mice

Obesity occurs when a person's calorie intake exceeds the amount of energy burns, which may lead to pathologic growth of adipocytes and the accumulation of fat in the tissues. In this study, the effect and mechanism of pear pomace extracts on 3T3-L1 adipocyte differentiation and apoptosis of mature adipocytes were investigated. The effects of pear pomace extract on cell viability and the anti-adipogenic and proapoptotic effects were investigated via MTT assay, Oil red O staining, western blot analysis and apoptosis assay. 3T3-L1 preadipocytes were stimulated with DMEM containing 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 microg/ml insulin and 1 microM dexamethasone for differentiation to adipocytes. 3T3-L1 cells were cultured with PBS or water extract of pear pomace. Water extract of pear pomace effectively inhibited lipid accumulations and expressions of PPAR-gamma and C/EBPalpha in 3T3-L1 cells. It also increased expression of p-AMPK and decreased the expression of SREBP-1c and FAS in 3T3-L1 cells. The induction of apoptosis was observed in 3T3-L1 cells treated with pear pomace. These results indicate that pear pomace water extract inhibits adipogenesis and induces apoptosis of adipocytes and thus can be used as a potential therapeutic substance as part of prevention or treatment strategy for obesity.
1-Methyl-3-isobutylxanthine ; 3T3-L1 Cells ; Adipocytes* ; Adipogenesis* ; Apoptosis* ; Blotting, Western ; Burns ; Cell Survival ; Dexamethasone ; Insulin ; Obesity ; Pyrus* ; Sterol Regulatory Element Binding Protein 1 ; Water*

BACKGROUND: Small cell lung cancer (SCLC) transformation during epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment in lung cancer has been suggested as one of possible resistance mechanisms. METHODS: We evaluated whether SCLC transformation or neuroendocrine (NE) differentiation can be found in the cell line model. In addition, we also investigated its effect on responses to conventional chemotherapeutic drugs of the SCLC treatment. RESULTS: Resistant cell lines to various kinds of EGFR-TKIs such as gefitinib, erlotinib, CL-387,785 and ZD6474 with A549, PC-9 and HCC827 lung adenocarcinoma cell lines were established. Among them, two resistant cell lines, A549/GR (resistant to gefitinib) and PC-9/ZDR (resistant to ZD6474) showed increased expressions of CD56 while increased synaptophysin, Rb, p16 and poly(ADP-ribose) polymerase were found only in A549/GR in western blotting, suggesting that NE differentiation occurred in A549/GR. A549/GR cells were more sensitive to etoposide and cisplatin, chemotherapeutic drugs for SCLC, compared to parental cells. Treatment with cAMP and IBMX induced synaptophysin and chromogranin A expression in A549 cells, which also made them more sensitive to etoposide and cisplatin than parental cells. Furthermore, we found a tissue sample from a patient which showed increased expressions of CD56 and synaptophysin after development of resistance to erlotinib. CONCLUSION: NE differentiation can occur during acquisition of resistance to EGFR-TKI, leading to increased chemosensitivity.
1-Methyl-3-isobutylxanthine ; Adenocarcinoma ; Blotting, Western ; Cell Line ; Cell Transformation, Neoplastic ; Chromogranin A ; Cisplatin ; Drug Resistance, Neoplasm ; Epidermal Growth Factor ; Etoposide ; Humans ; Lung ; Lung Neoplasms ; Parents ; Piperidines ; Poly(ADP-ribose) Polymerases ; Protein-Tyrosine Kinases ; Quinazolines ; Receptor, Epidermal Growth Factor ; Small Cell Lung Carcinoma ; Synaptophysin ; Erlotinib Hydrochloride

BACKGROUND: S-methylmethionine sulfonium chloride was originally called vitamin U because of its inhibition of ulceration in the digestive system. Vitamin U is ubiquitously expressed in the tissues of flowering plants, and while there have been reports on its hypolipidemic effect, its precise function remains unknown. OBJECTIVE: This study was designed to evaluate the anti-obesity effect of vitamin U in 3T3-L1 pre-adipocyte cell lines. METHODS: We cultured the pre-adipocyte cell line 3T3L1 to overconfluency and then added fat differentiation-inducing media (dexamethasone, IBMX [isobutylmethylxanthine], insulin, indomethacin) and different concentrations (10, 50, 70, 90, 100 mM) of vitamin U. Then, we evaluated changes in the levels of triglycerides (TGs), glycerol-3-phosphate dehydrogenase (G3PDH), AMP-activated protein kinase (AMPK), adipocyte-specific markers (peroxisome proliferator-activated receptor gamma [PPAR-gamma], CCAAT/enhancer-binding protein alpha [C/EBP-alpha], adipocyte differentiation and determination factor 1 [ADD-1], adipsin, fatty acid synthase, lipoprotein lipase) and apoptosis-related signals (Bcl-2, Bax). RESULTS: There was a gradual decrease in the level of TGs, C/EBP-alpha, PPAR-gamma, adipsin, ADD-1 and GPDH activity with increasing concentrations of vitamin U. In contrast, we observed a significant increase in AMPK activity with increasing levels of vitamin U. The decrease in bcl-2 and increase in Bax observed with increasing concentrations of vitamin U in the media were not statistically significant. CONCLUSION: This study suggests that vitamin U inhibits adipocyte differentiation via down-regulation of adipogenic factors and up-regulation of AMPK activity.
1-Methyl-3-isobutylxanthine ; Adipocytes ; AMP-Activated Protein Kinases ; Cell Line ; Complement Factor D ; Digestive System ; Down-Regulation ; Fatty Acid Synthetase Complex ; Flowers ; Glycerolphosphate Dehydrogenase ; Insulin ; Lipoproteins ; Triglycerides ; Ulcer ; Up-Regulation ; Vitamin U ; Vitamins

Substantia gelatinosa (SG) neurons receive synaptic inputs from primary afferent Adelta- and C-fibers, where nociceptive information is integrated and modulated by numerous neurotransmitters or neuromodulators. A number of studies were dedicated to the molecular mechanism underlying the modulation of excitability or synaptic plasticity in SG neurons and revealed that second messengers, such as cAMP and cGMP, play an important role. Recently, cAMP and cGMP were shown to downregulate each other in heart muscle cells. However, involvement of the crosstalk between cAMP and cGMP in neurons is yet to be addressed. Therefore, we investigated whether interaction between cAMP and cGMP modulates synaptic plasticity in SG neurons using slice patch clamp recording from rats. Synaptic activity was measured by excitatory post-synaptic currents (EPSCs) elicited by stimulation onto dorsal root entry zone. Application of 1 mM of 8-bromoadenosine 3,5-cyclic monophosphate (8-Br-cAMP) or 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) for 15 minutes increased EPSCs, which were maintained for 30 minutes. However, simultaneous application of 8-Br-cAMP and 8-Br-cGMP failed to increase EPSCs, which suggested antagonistic cross-talk between two second messengers. Application of 3-isobutyl-1-methylxanthine (IBMX) that prevents degradation of cAMP and cGMP by blocking phosphodiesterase (PDE) increased EPSCs. Co-application of cAMP/cGMP along with IBMX induced additional increase in EPSCs. These results suggest that second messengers, cAMP and cGMP, might contribute to development of chronic pain through the mutual regulation of the signal transduction.
1-Methyl-3-isobutylxanthine ; Adenosine ; Animals ; Chronic Pain ; Guanosine ; Myocytes, Cardiac ; Neurons ; Neurotransmitter Agents ; Plastics ; Rats ; Second Messenger Systems ; Signal Transduction ; Spinal Nerve Roots ; Substantia Gelatinosa

BACKGROUNDAdipose-derived stromal cell (ADSC) differentiation into neural cells in vitro is becoming widely studied. However, there are few reports on astrocytes following differentiation, and particularly on maturation and electrophysiology. In this study, we used various methods to determine ADSC-derived astrocyte maturity.

METHODSChemical induction with isobutylmethylxanthine (IBMX) was used to differentiate adult ADSCs into astrocytes followed by hematoxylin-eosin (HE) staining to observe morphology and transmission electron microscopy for cellular ultrastructure assessment. Immunofluorescence was used to detect expression of neural stem cell marker nestin as well as glial markers glial fibrillary acidic protein (GFAP) and S-100. In addition, we measured membrane potentials in bis-(1,3-dibarbituric acid) trimethine oxanol-labeled ADSCs and astrocytes by stimulation with a high potassium solution under an inverted fluorescence microscope. Finally, cell cycle distribution was detected by flow cytometry.

RESULTSTypical astrocyte morphology was shown by HE staining after 48-hour differentiation. Glial fibril was observed with transmission electron microscopy. GFAP and S-100 were not expressed in the control group, but were expressed within 24-hour differentiation and reached a maximum at day 14 with no change up to day 28. Nestin was weakly expressed in control cells and also reached a maximum at day 14 with the percentage of positive cells constant until day 21 followed by a decrease. Differentiated cell membrane potentials after stimulation with potassium were slightly increased, and then gradually declined over time. There was no significant membrane potential change in the control group. Flow cytometry showed that the percentage of cells in G0/G1 phase was 93% and only 5% in S phase.

CONCLUSIONADSCs were differentiated into mature astrocytes with typical characteristics including morphology, ultrastructure, marker protein expression, mature potassium channels and mitotic capacity.

1-Methyl-3-isobutylxanthine ; pharmacology ; Adipose Tissue ; cytology ; Adult ; Astrocytes ; cytology ; Barbiturates ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Electrophysiology ; methods ; Female ; Flow Cytometry ; Glial Fibrillary Acidic Protein ; metabolism ; Humans ; Male ; Membrane Potentials ; drug effects ; Microscopy, Fluorescence ; S100 Proteins ; metabolism ; Stromal Cells ; cytology ; Young Adult

Xanthohumol (XH), the principal prenylflavonoid of the hop plant (Humulus lupulus L.), dose-dependently inhibited isobutylmethylxanthine (IBMX)-induced melanogenesis in B16 melanoma cells, with little cytotoxicity at the effective concentrations. Decreased melanin content was accompanied by reduced tyrosinase enzyme activity, protein and mRNA expression. The levels of tyrosinase-related protein 1 and 2 mRNAs were decreased by XH. XH also inhibited alpha-melanocyte stimulating hormone- or forskolin-induced increases in melanogenesis, suggesting an action on the cAMP-dependent melanogenic pathway. XH downregulated the protein and mRNA expression of microphthalmia-associated transcription factor (MITF), a master transcriptional regulator of key melanogenic enzymes. These results suggest that XH might act as a hypo-pigmenting agent through the downregulation of MITF in the cAMP-dependent melanogenic pathway.
1-Methyl-3-isobutylxanthine/pharmacology ; Animals ; Cell Line ; Cell Survival/drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; Drug Antagonism ; Forskolin/pharmacology ; *Humulus ; Intramolecular Oxidoreductases/antagonists & inhibitors/biosynthesis ; Melanins/antagonists & inhibitors/*biosynthesis ; Melanocytes/*drug effects/*metabolism ; Melanoma, Experimental ; Membrane Glycoproteins/antagonists & inhibitors/biosynthesis ; Mice ; Microphthalmia-Associated Transcription Factor/antagonists & inhibitors ; Monophenol Monooxygenase/antagonists & inhibitors/biosynthesis/genetics ; Oxidoreductases/antagonists & inhibitors/biosynthesis ; Propiophenones/*pharmacology ; Signal Transduction/drug effects ; alpha-MSH/metabolism

AIMTo clarify the relationship between bicarbonate and cAMP in the promoting effects on the sperm agglutination.

METHODSSpermatozoa were collected from mature boars, washed and resuspended in a modified Krebs-Ringer HEPES lacking calcium chloride (mKRH). The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 min and then the percentage of head-to-head agglutinated spermatozoa was determined.

RESULTSSupplementation of the mKRH with sodium bicarbonate (5-10 mM) significantly raised the percentage of head-to-head agglutinated spermatozoa in the samples. The addition of selective inhibitors for calcium/calmodulin-dependent phosphodiesterases (type 1: 8-methoxymethyl-IBMX and vinpocetine, 25-50 micro M) or for cAMP-specific phosphodiesterases (type 4: Ro20-1724 and rolipram, 25-50 microM) enhanced the effect of bicarbonate on sperm agglutination as highly as did the addition of non-selective inhibitors for phosphodiesterases (IBMX and papaverine, 25-50 microM). A calmodulin antagonist (W-7, 2 microM), that potentially blocks the stimulator of the calcium/calmodulin-dependent phosphodiesterases, significantly enhanced the effect of bicarbonate on sperm agglutination. Moreover, a phosphodiesterase-resistant cAMP analogue (cBiMPS, 0.1 mM) markedly induced agglutination in more spermatozoa (76%) after the incubation without bicarbonate and phosphodiesterase inhibitors than did a less potent cAMP analogue (dibutyryl cAMP, 1 mM) (21%), while three kinds of cGMP analogues (0.1-1 mM) had no effect on sperm agglutination. In addition, a cAMP antagonist (Rp-cAMPS, 1 mM) significantly reduced the sperm agglutination resulting from the actions of bicarbonate and IBMX. On the other hand, the effect of bicarbonate was abolished by a change of incubation temperature from 38.5 degrees C to 25 degrees C.

CONCLUSIONThese findings demonstrate that the bicarbonate-induced agglutination of boar spermatozoa is controlled via the cAMP-mediated, temperature-dependent signaling cascade. This cascade is suppressed by the action of the phosphodiesterase (at least types 1 and 4).

1-Methyl-3-isobutylxanthine ; pharmacology ; Animals ; Bucladesine ; pharmacology ; Cyclic AMP ; physiology ; Cyclic GMP ; analogs & derivatives ; pharmacology ; physiology ; Male ; Papaverine ; pharmacology ; Purinergic P1 Receptor Antagonists ; Sodium Bicarbonate ; pharmacology ; Sperm Agglutination ; drug effects ; physiology ; Sperm Head ; drug effects ; physiology ; Swine ; Theophylline ; analogs & derivatives ; pharmacology

OBJECTIVETo investigate the molecular mechanism of human ether-a-go-go-related gene (HERG) potassium channels regulated by protein kinase A (PKA) in a human cell line.

METHODSHERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, and currents were measured with the patch clamp technique. The direct phosphorylation of HERG channel proteins expressed heterologously in Xenopus laevis oocytes was examined by (32)P labeling and immunoprecipitation with an anti-HERG antibody.

RESULTSElevation of the intracellular cAMP-concentration by incubation with the adenylate cyclase activator, forskolin (10 micromol/L), and the broad range phosphodiesterase inhibitor, IBMX (100 micromol/L), caused a HERG tail current reduction of 83.2%. In addition, direct application of the membrane permeable cAMP analog, 8-Br-cAMP (500 micromol/L), reduced the tail current amplitude by 29.3%. Intracellular application of the catalytic subunit of protein kinase A (200 U/ml) led to a tail current decrease by 56.9% and shifted the activation curve by 15.4 mV towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of approximately 155 kDa and a lower band with a molecular mass of approximately 135 kDa, indicating that both the core- and the fully glycosylated forms of the protein were phosphorylated.

CONCLUSIONSPKA-mediated phosphorylation of HERG channels causes current reduction in a human cell line. The coupling between the repolarizing cardiac HERG potassium current and the protein kinase A system could contribute to arrhythmogenesis under pathophysiological conditions.

1-Methyl-3-isobutylxanthine ; pharmacology ; 8-Bromo Cyclic Adenosine Monophosphate ; pharmacology ; Adenylyl Cyclases ; metabolism ; Animals ; Anti-Arrhythmia Agents ; pharmacology ; Cation Transport Proteins ; Cell Line ; Colforsin ; pharmacology ; Cyclic AMP ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; DNA-Binding Proteins ; ERG1 Potassium Channel ; Enzyme Activation ; drug effects ; Ether-A-Go-Go Potassium Channels ; Female ; Humans ; Membrane Potentials ; drug effects ; Microinjections ; Oocytes ; Patch-Clamp Techniques ; Phenethylamines ; pharmacology ; Phosphodiesterase Inhibitors ; pharmacology ; Phosphoric Diester Hydrolases ; drug effects ; metabolism ; Phosphorylation ; Potassium Channels ; genetics ; metabolism ; physiology ; Potassium Channels, Voltage-Gated ; RNA, Complementary ; administration & dosage ; genetics ; Sulfonamides ; pharmacology ; Trans-Activators ; Transcriptional Regulator ERG ; Xenopus laevis