• Clinical and Experimental Reproductive Medicine
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• 제25권1호
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• pp.77-86
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• 1998

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free T6 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at l20hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUT1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture ($93.8{\pm}3.1$, n=35) is significantly higher than that of control and glucose-free group ($76.6{\pm}3.8$, n=35 and $68.2{\pm}4.3$, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

• 고려인삼학회:학술대회논문집
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• 고려인삼학회 2007년도 춘계 학술대회
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• pp.23-24
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• 2007

Purpose: 인삼이 항당뇨 활성을 가진다는 연구가 많은 연구자들에 의해 진행되었고, 이는 인삼의 구성 성분 중 ginsenoside에 기인한다는 보고가 있다. 본 연구는 ginsenoside의 항당뇨 작용기전을 in vitro에서 알아보고자 3T3-L1 지방세포에서 glucose uptake와 췌장 베타세포인 HIT-T15 세포에서 insulin 분비 효과를 확인하였다. 이를 위하여 인삼을 식초로 처리한 긴삼의 70% MeOH 분획으로부터 protopanaxadiol 계인 ginsenoside $Rb_2$, $Rg_3$ 그리고 protopanaxtriol 계인 $Rg_2$를 분리하여 본 실험에 사용하였다. Method: Ginsenoside $Rb_2$, $Rg_2$, $Rg_3$가 지방 세포에서 glucose uptake에 미치는 효과를 확인하기 위하여 3T3-L1 세포를 DMEM (Dulbecco's Modified Eagle's Medium) 배지에서 분화 유도시켰으며 3T3-L1 preadipocyte가 80% 정도 자라면 분화 유도 배지 (5% fetal bovine serum (FBS), 0.5 mM isobutylmethylxanthine (IBMX), 1 mM dexamethasone 그리고 $10{\mu}g/ml$ insulin가 포함된 DMEM)로 4일, $10{\mu}g/ml$ insulin가 포함된 DMEM으로 2일, FBS만 포함된 DMEM으로 2일 배양하여 총 8일 동안 분화를 유도하였다. 분화 유도된 3T3-L1 adipocytes 에 각각 $Rb_2$, $Rg_2$, $Rg_3$를 $20{\mu}M$로 처리하여 16시간 배양하여 low glucose DMEM에서 3시간 배양한 후에 $37^{\circ}C$에서 insulin 10 ng/ml 과 각각 $Rb_2$, $Rg_2$, $Rg_3$가 포함된 Krebs Ringer Hepes buffer(KRP buffer)에서 20분간 배양하였다. 2-deoxy-D-[$^3H$]-glucose를 넣고 10분 후에 차가운 PBS로 반응을 종결시켜 lysis buffer로 cell을 모은 후 scintillation counter를 이용하여 glucose를 측정하였다. Insulin 분비 효과는 HIT-T15 세포와 일차 배양한 흰쥐 소도세포(islets)를 사용하여 확인하였다. HIT-T15 세포는 24 well plate에 well 당 $2{\times}10^5$ 개씩 분주하여 24시간 동안 배양한 후 시료를 처리하였으며 소도 세포는 Sprague-Dawley rat의 췌장에 collagenase가 포함된 Hanks' Balanced Salt Solution(HBSS)을 주입하여 분리하고 islets을 얻었다. 분리한 소도세포를 $1{\sim}2$일 동안 배양하여 $Rb_2$, $Rg_2$, $Rg_3$가 각각 $20{\mu}M$의 농도로 첨가된 insulin 측정용 buffer인 Krebs-Ringer buffer (KRB+0.3% BSA, KRBB)에 $37^{\circ}C$에서 1시간 incubation 시킨 후 배양액으로 분비된 인슐린의 양을 측정하였다. 한편 ginsenoside의 인슐린 분비 촉진 기전을 알아보기 위한 실험에서는 ATP-sensitive $K^+$ channel opener인 diazoxide (0.5 mM)가 ginsenoside에 의해 촉진된 인슐린 분비를 억제하는지 살펴보았다. Result: glucose uptake assay 에서는 $Rg_2$가 가장 크게 glucose uptake를 증가시켰고 $Rb_2$, $Rg_3$는 그 활성이 크지 않았다. 한편 Insulin 분비 효과는 diol계인 $Rg_3$에서 용량 의존적으로 인슐린의 분비를 촉진시켰으며 $20{\mu}M$ 농도에서 대조군과 비교해 1.5배 이상의 분비 촉진 효과를 보였고 triol계인 $Rg_2$ 에서는 이러한 효과가 나타나지 않았다. $Rg_3$의 인슐린 분비 촉진 기전을 0.5 mM 의 diazoxide를 이용하여 확인한 결과 $Rg_3$에 의해 촉진된 인슐린 분비를 감소시켰다. 이로 미루어보아 $Rg_3$의 인슐린 분비 촉진 기전은 ATP-sensitive $K^+$ 채널의 봉쇄에 의한 것임을 확인할 수 있었다.

• 미생물학회지
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• 제13권2호
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• pp.45-50
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• 1975

Uptake of $^{14}C$-sorbose and $^{14}C$-3-O-methylglucose by ungerminated conidia of Neurospora crassa was measured by means of the millipore filter technique. Initial rates of jptake of both sorbose and 3-O-methylglucose show a marked dependence optimal pH for uptake of both sugars is close to 4.75. When ungerminated conidia are "starved" with buffer for a prolonged period of time prior to assaying their transport capacity and mycelia, no de-repression of the glucose-repressible sugar transport system is effectuated in contrast to the findings for germinated conidia.d conidia.

• 약학회지
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• 제40권2호
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• pp.218-224
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• 1996

This laboratory has recently reported the synthesis and in vitro antitumor activity of PT(II) complexes containing ethylenediamine and diphosphine. In view of the reports of others, cisplatin is toxic to the kidney since the kidney's vulnerability to PT(II) complexes may originate in its ability to accumulate and retain platinum to a greater degree than other organs. The in vitro cytotoxicity of these synthetic PT(II) complexes on the primary cultured proximal tubular cells of rabbit kidney and renal cortical cells of human kidney was investigated. Three endpoints for cytotoxicity tests were evaluated:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), $^3H$-thymidine uptake and the glucose consumption tests. The rank order of sensitivity exhibited $^3H$-thymidine uptake>MTT>glucose consumption test. The agents with diphosphine leaving group were significantly less cytotoxic than cisplatin. Moreover, 1,2-bis(diphenylphosphino)ethane (DPPE) exhibited less cytotoxicity than 1.3-bis (diphenylphosphino)propane (DPPP) against on rabbit and human cultured kidney cells. Based on these results, the decreased nephrotoxicity of these new complexes over cisplatin appeared to be partially attributable to a leaving group of DPPP and DPPE. This novel class of platinum compound represents a valuable lead in the development of a "third-generation" agent.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.248-254
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• 2008

Candida magnoliae, an osmotolerant and erythritol producing yeast, prefers D-fructose to D-glucose as carbon sources. For the investigation of the fructophilic characteristics with respect to sugar transportation, a sequential extraction method using various detergents and ultracentrifugation was developed to isolate cellular membrane proteins in C. magnoliae. Immunoblot analysis with the Pma1 antibody and two-dimensional electrophoresis analysis coupled with MS showed that the fraction II was enriched with membrane proteins. Eighteen proteins out of 36 spots were identified as membrane or membrane-associated proteins involved in sugar uptake, stress response, carbon metabolism, and so on. Among them, three proteins were significantly upregulated under the fructose supplying conditions. The hexose transporter was highly homologous to Ght6p in Schizosaccharomyces pombe, which was known as a predominant transporter for the fructose uptake of S. pombe because it exhibited higher affinity to D-fructose than D-glucose. The physicochemical properties of the ATP-binding cassette transporter and inorganic transporter explained their direct or indirect associations with the fructophilic behavior of C. magnoliae. The identification and characterization of membrane proteins involved in sugar uptake might contribute to the elucidation of the selective utilization of fructose to glucose by C. magnoliae at a molecular level.

• 대한한의학회지
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• 제23권1호
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• pp.83-91
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• 2002

Objectives : In this study, water extracts from Coix lachryma-jobi Linne var. mayuen Stapf. were investigated for their effects on insulin-like action and glucose uptake in 3T3-Ll cells. Methods : We examined the effects of insulin-like action on the differentiation of 3T3-Ll fibroblasts. The Coicis Semen was treated with hot water and the extract was freeze-dried. The hot water extract was chromatographed on nonionic polymer resin (Amberlite XAD-4, Sigma) with distilled water (Fr. 1), 20% (Fr. 2), 40% (Fr. 3), 60% (Fr. 4), 80% (Fr. 5), and 100% EtOH (Fr. 6), successively. Results : Total extract of Coicis Semen was fractionated into 0 to 100% MeOH with Amberlite XDA-4 column. Treatment of cells with $10{\;}\mu\textrm{g}/ml$ of total extracts of Coicis Semen significantly increased the differentiation (p<0.05). At $1{\;}\mu\textrm{g}/ml$ level of insulin, the differentiation was accelerated (p<0.01). The effect of extracts plus insulin on the differentiation was greater than that of insulin alone. In 3T3-Ll adipocytes, glucose uptake was higher by 2.7 times with 5 uM of total extract in low dosage of insulin (3 ng/ml) than without total extract. 5 and 50 uM of water and 40% MeOH fractions increased glucose uptake by 3.5 times in 3T3-Ll adipocytes (p<0.00l). Conclusions : Coicis Semen contains compounds which playa role of insulin-like action and insulin sensitizer.

• Biomolecules & Therapeutics
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• 제2권1호
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• pp.65-70
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• 1994

Hypoglycemic mechanism of brazilin was investigated in the streptozotocin induced diabetic rats. Plasma glucose levels of diabetic rats were significantly ame]iorated by the treatment of brazilin, but there were no changes in plasma insulin levels. Brazilin increased insulin binding capacity to epididymal adipocytes, and Scatchard analysis revealed that this increase in insulin binding was not due to the increase of insulin binding sites but that of binding affinity. 2-Deoxyglucose uptake of epididymal adipocytes was significantly augmented by the intraperitoneal administration of brazilin and the same result was obtained in in vitro study. Glucose oxidation and lipogenesis in epididymal adipocytes were significantly enhanced by the treatment of bracilin.

• 대한약리학회지
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• 제10권1호
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• pp.55-59
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• 1974

Further elucidation of the mechanism of halothane's negative inotropic action has resulted from a study of the effect of various substrates on halothane-depressed rat atria. Approximately 6 mg% halothane was required to maintain a 50% depression of the contractility of rat atria suspended in a modified Krebs-Ringer bicarbonate glucose medium, pH 7.4, $30^{\circ}C$ for 2hr. Both lactate and acetate were found to restore partially the contractility of halothane-depressed atria. The maximally effective concentration of lactate was 5 mM; for acetate it was 2.5mM. Neither 5 nor 20 mM of additional glucose was effective in restoring the force of contraction of halothane-depressed atria. The results are consistent with the hypothesis that halothane exerts at least a part of its negative inotropic effect on rat atria by inhibiting either the uptake or utilization of glucose by the myocardium. The site of blockade must be prior to the conversion of pyruvate to acetyl CoA. In our previous report dealing with the mechanism of cardiac depressant action of inhalation anesthetic halothane, it has been demonstrated that: 1) approximately 6 mg/100 ml halothane is required to maintain 50% depression of the force of contraction of isolated rat atria in Krebs-Ringer bicarbonate glucose medium; 2) pyruvate partially restores the contractility of halothane-depressed atria, but has no effect on normal atria; the partial recovery of depressed atria by the addition of sodium pyruvate is due to the effect of the pyruvate ion itself, not to the sodium ion; 4) addition of pyruvate, to atria depressed with hypertonic medium, produced only further depression. From these findings we concluded that the cardiac depressant action of halothane on rat atria is a manifestation of inhibition of glucose uptake or utilization. The present studies were undertaken to observe the effect of other substrates on halothane-depressed atria in order to substantiate our conclusion. As with the case of pyruvate, lactate and acetate also partially restored the force of contraction of halothane-depressed atria. These data are consistent with the hypothesis that halothane inhibits glucose uptake or utilization in the glycolytic cycle of the myocardium.

• 한국식품저장유통학회지
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• 제24권7호
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• pp.1007-1016
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• 2017

다슬기는 예로부터 간염, 간경화, 지방간 등의 치료 및 개선에 이용되어 왔으며, 특히 소변불통, 소갈증(당뇨) 등의 약용으로 이용되어 왔다. 본 연구에서는 이러한 다슬기를 대상으로 항당뇨에 대한 효능을 과학적으로 검증하고 그 기작을 규명하고자 하였다. 먼저 다슬기의 생물학적 기능성을 높이기 위해 효소 가수분해를 실시하였으며, protamex에 의한 가수분해도는 10시간 후 약 43% 수준을 나타내었다. PTP1B는 인슐린 신호전달기전에서 IRS-1의 인산화를 방해하여 인슐린 민감성을 저해시키는 효소이다. protamex를 이용한 다슬기 가수분해물(MPH)의 PTP1B에 대한 저해활성은 $15.42{\pm}1.1{\mu}g/mL$의 $IC_{50}$ 값을 나타내어 양성대조군 ursolic acid의 $16.7{\mu}g/mL$ 보다 높은 저해활성을 보이면서 강한 항당뇨 활성 소재로서의 가능성을 보였다. 이에 따라 유리지방산을 이용하여 C2C12 myoblast에서 인슐린 저항성을 유도하고, MPH에 의한 포도당 흡수 정도를 확인하였다. 그 결과, 1 mM PA 처리에 의해 약 32% 수준으로 떨어진 포도당 흡수율은 MPH 처리에 의해 약 199% 수준으로 증가하였다. 또한 장기간 고농도의 포도당(30 mM)에 의해 유도된 당독성 조건에서 MPH는 췌장의 베타세포 INS-1 세포의 생존율을 증가시키고, 대조군에 비해 약 160% 인슐린 mRNA 발현량을 증가시켰다. 이러한 결과에서 MPH는 PTP1B 활성을 저해함으로써 인슐린 신호전달 기작을 활성화하고, 인슐린저항성 환경에서 포도당 흡수를 증진시켜 인슐린저항성을 개선하며, 나아가 고농도 포도당에 의해 유도되는 당독성환경에서 췌장 베타세포를 보호하고 인슐린 mRNA발현량을 정상화할 수 있다는 것을 확인할 수 있었다.

• 동의생리병리학회지
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• 제22권2호
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• pp.340-345
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• 2008

Extract of Acanthopanax senticosus has recently been demonstrated to possess significant antidiabetic potential, in accordance with the traditional use of this plant as an antidiabetic natural health product. The present study evaluated the effects of fermented extract (FE) of this plant on glucose-stimulated insulin secretion, glucose uptake, and streptozotocin-induced type 1 diabetes model. A 3 h pretreatment with FE prevented $IL-1{\beta}$ and $IFN-{\gamma}$ toxicity in isolated rat islets. However, it did not affect insulin-stimulated glucose uptake in C2C12 myotubes. In addition, pretreatment of mice with FE blocked the destruction of streptozotocin-induced islets and the development of type 1 diabetes. FE reduced blood glucose level, increased insulin secretion, and improved glucose tolerance in streptozotocin-treated mice, whereas nonfermented extract (NFE) had moderate effects. Immunohistochemical staining for insulin clearly showed that pretreatment with FE blocked the STZ-induced islets destruction and restored the number of islet cells that secreted insulin to the level of the control. Although the active principles and their mechanisms of action remain to be identified, FE may nevertheless represent a novel complementary therapy and a source of novel therapeutic agents against type 1 diabetes mellitus.