• The Korean Journal of Physiology
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• v.30 no.1
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• pp.63-76
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• 1996

The aim of present study was to characterize phosphate uptake and to investigate the mechanism for the insulin and insulin-like growth factor(IGF) stimulation of phosphate uptake in primary cultured rabbit renal proximal tubule cells. Results were as follows : 1. The primary cultured proximal tubule cells had accumulated $6.68{\pm}0.70$ nmole phosphate/mg protein in the presence of 140 mM NaCl and $2.07{\pm}0.17$ nmole phosphate/mg protein in the presence of 140 mM KCl during a 60 minute uptake period. Raising the concentration of extracellular phosphate to 100 mM$(48.33{\pm}1.76\;pmole/mg\;protein/min)$ induced decrease in phosphate uptake compared with that in control cells maintained in 1 mM phosphate$(190.66{\pm}13.01\;pmole/mg\;protein/min)$. Optimal phosphate uptake was observed at pH 6.5 in the presence of 140 mM NaCl. Phosphate uptake at pH 7.2 and pH 7.9 decreased to $83.06{\pm}5.75%\;and\;74.61{\pm}3.29%$ of that of pH 6.5, respectively. 2. Phosphate uptake was inhibited by iodoacetic acid(IAA) or valinomycin treatment $(62.41{\pm}4.40%\;and\;12.80{\pm}1.64%\;of\;that\;of\;control,\;respectively)$. When IAA and valinomycin were added together, phosphate uptake was inhibited to $8.04{\pm}0.61%$ of that of control. Phosphate uptake by the primary proximal tubule cells was significantly reduced by ouabain treatment$(80.27{\pm}6.96%\;of\;that\;of\;control)$. Inhibition of protein and/or RNA synthesis by either cycloheximide or actinomycin D markedly attenuated phosphate uptake. 3. Extracellular CAMP and phorbol 12-myristate 13 acetate(PMA) decreased phosphate uptake in a dose-dependent manner in all experimental conditions. Treatment of cells with pertussis toxin or cholera toxin inhibited phosphate uptake. cAMP concentration between $10^{-6}\;M\;and\;10^{-4}\;M$ significantly inhibited phosphate uptake. Phosphate uptake was blocked to about 25% of that of control at 100 ng/ml PMA. 3-Isobutyl-1-methyl-xanthine(IBMX) inhibited phosphate uptake. However, in the presence of IBMX, the inhibitory effect of exogenous cAMP was not significantly potentiated. Forskolin decreased phosphate transport. Acetylsalicylic acid did not inhibit phosphate uptake. The 1,2-dioctanoyl-sn-glycorol(DAG) and 1-oleoyl-2-acetyl-sn- glycerol(OAG) showed a inhibitory effect. However, staurosporine had no effect on phosphate uptake. When PMA and staurosporine were treated together, inhibition of phosphate uptake was not observed. In conclusion, phosphate uptake is stimulated by high sodium and low phosphate and pH 6.5 in the culture medium. Membrane potential and intracellular energy levels are also an important factor fer phosphate transport. Insulin and IGF-I stimulate phosphate uptake through a mechanisms that involve do novo protein and/or RNA synthesis and decrease of intracellular cAMP level. Also protein kinase C(PKC) is may play a regulatory role in transducing the insulin and IGF-I signal for phosphate transport in primary cultured proximal tubule cells.

• Nutrition Research and Practice
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• v.12 no.6
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• pp.494-502
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• 2018

BACKGROUND/OBJECTIVES: Reducing the number of adipocytes by inducing apoptosis of mature adipocytes as well as suppressing differentiation of preadipocytes plays an important role in preventing obesity. This study examines the anti-adipogenic and pro-apoptotic effect of red pepper seed water extract (RPS) prepared at $4^{\circ}C$ (RPS4) in 3T3-L1 cells. MATERIALS/METHODS: Effect of RPS4 or its fractions on lipid accumulation was determined in 3T3-L1 cells using oil red O (ORO) staining. The expressions of AMP-activated protein kinase (AMPK) and adipogenic associated proteins [peroxisome proliferator-activated receptor-${\gamma}$ ($PPAR-{\gamma}$), CCAAT/enhancer-binding proteins ${\alpha}$ (C/EBP ${\alpha}$), sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC)] were measured in 3T3-L1 cells treated with RPS4. Apoptosis and the expression of Akt and Bcl-2 family proteins [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated death promoter (Bad), Bcl-2 like protein 4 (Bax), Bal-2 homologous antagonist/killer (Bak)] were measured in mature 3T3-L1 cells treated with RPS4. RESULTS: Treatment of RPS4 ($0-75{\mu}g/mL$) or its fractions ($0-50{\mu}g/mL$) for 24 h did not have an apparent cytotoxicity on pre and mature 3T3-L1 cells. RPS4 significantly suppressed differentiation and cellular lipid accumulation by increasing the phosphorylation of AMPK and reducing the expression of $PPAR-{\gamma}$, C/EBP ${\alpha}$, SREBP-1c, FAS, and ACC. In addition, all fractions except ethyl acetate fraction significantly suppressed cellular lipid accumulation. RPS4 induced the apoptosis of mature adipocytes by hypophosphorylating Akt, increasing the expression of the pro-apoptotic proteins, Bak, Bax, and Bad, and reducing the expression of the anti-apoptotic proteins, Bcl-2 and p-Bad. CONCLUSIONS: These finding suggest that RPS4 can reduce the numbers as well as the size of adipocytes and might useful for preventing and treating obesity.

• The Korea Journal of Herbology
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• v.29 no.2
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• pp.15-21
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• 2014

Objectives : The purpose of this study was to investigate inhibitory effects of steamed Polygonatum odoratum extract (POE) on differentiation and adipogenesis in 3T3-L1 adipocytes. Methods : Polygonatum odoratum (P. odoratum) extract was extracted with ethyl acetate. Total phenolic and flavonoid contents in POE were measured for antioxidant activity. The spectrophotometric method was used to determine the DPPH and ABTS radical scavenging activity and ferric-reducing antioxidant potential (FRAP). MTT assay was examined for cell toxicity, oil red O staining was performed for intracelluar adipogenesis in differentiated 3T3-L1 adipocytes. Western blot analysis for measurement of CCAAT/enhancer-binding protein ${\alpha}$ ($C/EBP{\alpha}$), peroxisome proliferator-activated receptor${\gamma}$ ($PPAR{\gamma}$) and AMP-activated protein kinase (AMPK) expressions were performed. Results : The results revealed that POE has antioxidant activities. Contents of total polyphenolics and flavonoids were $50.83{\pm}1.52$ GAE mg/100g dry weight of POE and $17.05{\pm}2.47$ RE mg/100g dry weight of POE, respectively. DPPH radical scavenging activity, and FRAP in 10 mg/ml concentration were $92.1{\pm}0.6%$, $244.8{\pm}9.0{\mu}M$ Fe(II) and ABTS inhibition in 5 mg/ml concentration was $84.8{\pm}4.1%$. Treatment of POE in adipocytes inhibited the differentiation and adipogenesis of 3T3-L1 adipocytes compared to those of vehicle control. Additionally, protein expressions of $C/EBP{\alpha}$ and $PPAR{\gamma}$, major transcription factor for the adipogenic genes, were significantly decreased compared to those of vehicle control (p<0.05). Futhermore, phosphorylation of AMPK was increased in 3T3-L1 adipocytes treated with POE compared to that of vehicle control (p<0.05). Conclusions : we demonstrate that steamed P. odoratum extract (POE) has potentiating antioxidant activities, inhibits differentiation and lipid accumulation and also induces energy expenditure in adipocytes, which may contribute to antiobesity property.

• The Korea Journal of Herbology
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• v.29 no.6
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• pp.117-123
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• 2014

Objectives : Sinhyowoldo-san (SHWDS) is said to be a traditional medicine used for shigellosis, abdominal pain, diarrhea. But mechanism of SHWDS mediated-modulation of immune function is not sufficiently understood. To ascertain the molecular mechanisms of SHWDS 70% EtOH extract on pharmacological and biochemical actions in inflammation, we researched the effect of pro-inflammatory mediators in phorbol-12-myristate-13-acetate (PMA)+ A23187-activated human mast cell line (HMC-1). Methods : In the present research, cell viability was measured by MTS assay. pro-inflammatory cytokine production was measured by performing enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), and western blot analysis to analyze the activation of mitogen-activated protein kinases (MAPKs), nuclear factor kappa-light-chain-enhancer of activated B cells ($NF-{\kappa}B$). The investigation focused on whether SHWDS inhibited the expressions of interleukin-6 (IL-6), interleukin-8 (IL-8), MAPKs and $NF-{\kappa}B$ in PMA+A23187-activated HMC-1 cells. Results : SHWDS has no cytotoxicity at measured concentration (50, 100, and $250{\mu}g/ml$). SHWDS ($250{\mu}g/ml$) inhibits pro-inflammatory cytokine expression in PMA+ A23187-activated HMC-1 cells. Moreover, SHWDS inhibited cyclooxygenase (COX)-2 expression. In activated HMC-1 cells, SHWDS suppressed phosphorylation of extracellular signal-regulated kinase (ERK 1/2) and c-jun N-terminal Kinase (JNK 1/2). Then, SHWDS suppressed activation of nuclear factor $NF-{\kappa}B$ in nuclear, degradation of IkB ${\alpha}$ in cytoplasm. Conclusions : We propose that SHWDS has an anti-inflammatory therapeutic potential, which may result from inhibition of ERK 1/2, JNK 1/2 phosphorylation and $NF-{\kappa}B$ activation, thereby decreasing the expression of pro-inflammatory genes.

• Tuberculosis and Respiratory Diseases
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• v.40 no.3
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• pp.223-235
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• 1993

Background: Superoxide anion which was produced by macrophage and neutrophil has a defensive role to kill invasive microorganisms and also an injurious role to produce self lung damage. Production of oxygen free radicals including superoxide is a main mechanism of acute lung injury caused by bacterial endotoxin. Endotoxin is known to activate alveolar macrophage to produce increased oxygen free radicals after the stimulation with various biological materials (priming effect). Calcium is a very important intracellular messenger in that cellular process of superoxide production. Method: This experiment was performed to elucidate the effects of endotoxin and calcium on superoxide production by phorbol myristate acetate-stimulated alveolar macrophage and the effect of verapamil on priming effect of endotoxin. Results: 1) Preincubation of macrophages with endotoxin (E. coli 055-B5) primed the cells to respond with increased superoxide production after the stimulation with PMA. Priming with endotoxin ($10^{-1}$ug/ml) produced a maximal enhancement of superoxide production (43%). 2) Verapamil could inhibit the superoxide production by PMA stimulated macrophage regardless of the presence of extracellular calcium. This means that the inhibitory effect of verapamil is caused by a mechanism independent of blocking calcium influx. 3) Verapamil could inhibit the priming effect of endotoxin on alveolar macrophage (from 30% increment to 13% increment) and could inhibit the superoxide production by PMA-stimulated macrophage preincubated with endotoxin. Conclusion: We concluded that verapamil could inhibit the superoxide production by PMA-stimulated rat alveolar macrophage and also inhibit the priming effect of endotoxin on alveolar macrophage. These inhibitory effects of verapamil could be one of the mechanisms of verapamil effects on endotoxin induced lung injury.

• Journal of Life Science
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• v.26 no.1
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• pp.34-41
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• 2016

Black chokeberries (scientific name Aronia melanocarpa) have been reported to have major effects due to anti-oxidant, anti-inflammatory, and anti-cancer capabilities. In this study, we investigated the anti- wrinkle effects of A. melanocarpa, including collagenase inhibition effects and their molecular biological mechanisms, such as oxidative stress-induced matrix metalloproteinase (MMP), mitogen-activated protein (MAP) kinase, and activator protein (AP)-1 expression and/or phosphorylation. In collagenase inhibition activity, the ethyl acetate fraction of black chokeberry (AE) was 77.2% at a concentration of 500 μg/ml, which was a significant result compared to that of Epigallocatechin gallate (positive control, 83.9% in 500 μg/ml). In the reactive oxygen species (ROS) assay, the AE produced 78% of ROS in 10 μg/ml and 70% of ROS in 75 μg/ml, which was a much lower percentage than the ROS production of H₂O₂-induced CCRF S-180II cells. In the MTT assay, cell viability was increased dose-dependently with AE in H₂O₂-induced cells. In protein expression by western blot assay, the AE suppressed the expression and phosphorylation of MMPs (MMP-1, -3, -9), MAPK (ERK, JNK, and p38), and AP-1 (c-Fos and c-Jun), and expressed the pro-collagen type I in H₂O₂-induced cells. These results suggest that black chokeberries have anti-wrinkle and collagen-production effects, and they may be used in applications for material development in the functional food and cosmetic industries.

• Radiation Oncology Journal
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• v.19 no.2
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• pp.171-180
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• 2001

Purpose : Expression of TIMP, intrinsic inhibitor of MMP, is regulated by signal transduction in response to genotoxins and is likely to be an important step in metastasis, angiogenesis and wound healing after ionizing radiation. Therefore, we studied radiation mediated TIMP expression and its mechanism in head and neck cancer cell lines. Materials and Methods : Human head and neck cancer cell lines established at Asan Medical Center were used and radiosensitivity $(D_0)$, radiation cytotoxicity and metastatic potential were measured by clonogenic assay, n assay and invasion assay, respectively. The conditioned medium was prepared at 24 hours and 48 hours after 2 Gy and 10 Gy irradiation and expression of TIMP protein was measured by Elisa assay with specific antibodies against human TIMP. hTIMP1 promoter region was cloned and TIMP1 luciferase reporter vector was constructed. The reporter vector was transfected to AMC-HN-1 and -HN-9 cells with or without expression vector Ras, then the cells were exposed to radiation or PMA, PKC activator. EMSA was peformed with oligonucleotide (-59/-53 element and SP1) of TIMP1 promoter. Results : $D_0$ of HN-1, -2, -3, -5 and -9 cell lines were 1.55 Gy, 1.8 Gy, 1.5 Gt, 1.55 Gy and 2.45 Gy respectively. n assay confirmed cell viability, over $94\%$ at 24hrs, 48hrs after 2 Gy irradiation and over 73% after 10 Gy irradiation. Elisa assay confirmed that cells secreted TIMP1, 2 proteins continuously. After 2 Gy irradiation, TIMP2 secretion was decreased at 24hrs in HN-1 and HN-9 cell lines but after 10 Gy irradiation, it was increased in all cell lines. At 48hrs after irradiation, it was increased in HN-1 but decreased in HN-9 cells. But the change in TIMP secretion by RT was mild. The transcription of TIMP1 gene in HN-1 was induced by PMA but in HN-9 cell lines, it was suppressed. Wild type Ras induced the TIMP-1 transcription by 20 fold and 4 fold in HN-1 and HN-9 respectively. The binding activity to -59/-53, AP1 motif was increased by RT, but not to SP1 motif in both cell lines. Conclusions : We observed the difference of expression and activity of TIMPs between radiosensitive and radioresistant cell line and the different signal transduction pathway between in these cell lines may contribute the different radiosensitivity. Further research to investigate the radiation response and its signal pathway of TIMPs is needed.