인슐린에 의한 GLUT4 전위과정에서 Rab Protein 및 Gh Protein의 역할에 관한 연구

하종식,고영현,이지희 梨花女子大學校 醫科大學 醫科學硏究所 1998 EMJ (Ewha medical journal) Vol.21 No.4

인슐린이 지방세포나 근육세포에서 포도당의 이동을 촉진시키는 기전은 포도당 운반체 단백질(GLUT4)을 세포 내부의 저장소로부터 세포막 전이를 일으키는데 있다. 그러나 아직 GLUT4의 전이과정이 확실히 밝혀지지 않다. 최근 포유동물에서 GLUT4의 전이과정이 확실히 밝혀져 있지 않다. 최근 포유동물에서 GTP-결합단백질들이 세포내부의 물질이동에 관련이 있을 것이라는 보고가 있어 이들 단백질들이 GLUT4의 전이를 일으키는 signal cascade에 관여하는지를 구명하기 위하여 본 실험을 시작하였다. 실험동물로는 Sprague-Dawley 쥐를 사용하였고 str-eptozotocin 으로 당뇨를 유발시킨 후 인슐린을 처치한 군과 처치하지 않은 군으로 나누어 4주간 사육하였으며 이들을 각각 경추이골로 의생시켜 epididymal 지방조직을 분리하였다. 이 조직으로부터 지방세포를 분리하고 plasma membrane(PM), high density microsome(HDM), low density microsome(LDM) 등의 분획으로 나누어 GLUT4, Rab4 및 Gα_h 의 항체와 immu-nobotting 하여 인슐린에 의한 이들의 분포변동을 측정하였다. 실험결과는 다음과 같다. 1) GLUT4와 Rab4는 지방세포막에 공존하고 있으나 Gα_h 는 발견되지 않았다. 2) GLUT4와 Rab4는 인슐린의 처치에 의하여 PM에 증가하였고 LDM과 HDM에서는 가소하였다. 3) GLUT4의 이동에 비하여 Rab4는 PM으로의 이동정도가 대단히 낮았다. 이로부터 Rab4는 인슐린의 자극에 의한 GLUT4의 전이과정에 관련이 있는 것으로 생각되고 그 기전은 인슐린에 의해 Rab4가 활성화 되면 인산화 과정을 동반하여 down stream의 signal cascade를 활성화하여 세포막의 GLUT4 의 전이를 측정하는 것으로 생각된다. Gh 단백질은 GLUT4의 포도당 이동전기에 관여하지 않는 것으로 보아 포도당이동기전 Ca^2+ 를 동반하는 세포내의 기전과는 관련이 없는 것으로 추측된다. Insulin stimulation of glucose transport in adipocytes results from the translocation of vesicles containing the GLUT4 glucose transporter from an intracellular pool to the plasma membrane. In mammalian cells a family of GTP-binding proteins has been implicated in the control of cellular trffic. Thus this study was planned to see whether G-proteins such as Rab, a small molecular mass G-protein and Gα_h, a large molecular mass G-protein are involved in insulin induced GLUT4 translocation process. Diabetic rats(Spraque-Dauley, 200-250g) were prepared by injection of streptozotocin(60mg/kg,IP) and treated with or without insulin(20U/rat) for 4 weeks. The purpose of the study is to elucidate a possible functional relationship between G-protein and the insulin-responsive GLUT-4 translocation by immunoblotting method from the subcellular fractions of adipocytes of epididymal tissues. As results Rab4 protein was coexisted in the membrane of GLUT4 immunoprecipitates of adipocyte total homogenates in normal rats, however Gα_h, could not be detected. The amount of GLUT4 at plasma membrane(PM) obtained from insulin treated rats were increased by 21. 35% compared to that of streptozotocin diabetic rats. The increase of Rab4 at the same plasma membrane was negligible. On the other hand, the amounts of GLUT4 and Rab4 at low density microsome(LDM) were decreased by 7.82% and 9.25%, respectively. These results show that Rab4 is co-localized with GLUT4 in an insulin-responsive intracellular compartment and Rab4 protein plays role in the action of insulin on the GLUT4 translocation but a large molecular G-protein, Gα_h is not involved in the GLUT4 translocation process.

Vanadate가 소장절편 및 소장세포막의 Vesicle에서 포도당 이동에 미치는 영향

하종식,김구자 梨花女子大學校 醫科大學 醫科學硏究所 1989 EMJ (Ewha medical journal) Vol.12 No.3

Vanadate has been reported to inhibit Na^+, K^+-ATPase of many cells. Since intestinal uptake of glucose is influenced by the enzyme, we studied the effects of varying concentrations of sodium vanadate(NaVO_3) on the glucose transport in everted sacs prepared from the segment of jejunum and upper part of ileum of rabbit. At concentrations of range 10 ^8M~10 ^4M vanadate decreased the mucosal-to-serosal flux(S/M ratio) of glucose to 4.8% -38.8% accompanying inhibition of Na^+, K^+ -ATPase activities of basolateral membranes and alkaline phosphatase activities of brush-border membranes purified from small intestinal epithelial cells. On the other hand, it was found that there were two cytochalasin B binding sites at the basolateral membranes, of which site I showed a dissociation constant of 1.8×10 exp(8)M with maximal binding capacity of 26 pmoles/㎎ protein and site Ⅱ showed a dissociation constant of 9.3×10 exp(7)M with maximal binding capacity of 140 pmoles/㎎ protein. Vanadate(10 ^4M) inhibits cytochalasin B binding at the both binding sites to 38.5% and 45.7% respectively. Vanadate also inhibited cytochalasin B binding at both ghost membranes and band 4.5(glucose carrier) protein purified from human red blood cell membranes. SDS-polyacrylamide gel electrophoresis(SDS-PAGE) showed that there existed a band 4.5 like protein in both basolateral membrane and brush-border membrane. Based on the above results the following conclusions are obtained: 1) Glucose is absorbed across the small intestinal epithelium by a two stage process. the first is sodium co-transport system of the brush-border membrane and the second is sodium-independent facilitated diffusion system of the basolateral membrane. 2) The driving force for the transcellular movement of glucose is mainly dependent on sodium concentration gradient across the brush-border membrane which is generated by Na^+, K^+ ATPase at the basolateral membrane and also dependent directly on the activity of glucose carrier which may exist at the basolateral membrane. 3) Vanadate inhibits glucose uptake across the intestinal epithelium by affecting on both the Na^+, K^+ ATPase and glucose carrier of the basolateral membrane.

미세먼지의 건강부담 추정과 불확실성

하종식 환경독성보건학회 2014 한국독성학회 심포지움 및 학술발표회 Vol.2014 No.5

인체 적혈구막 Band 4.5 단백질의 기능적인 분자구조

하종식,Hah, Jong-Sik 대한생리학회 1986 대한생리학회지 Vol.20 No.2

The functional molecular weight of band 4.5 polypeptide was measured by applying the classical target theory to radiation inactivation data of the cytochalasin B binding. Band 4.5 polypeptides purified from human erythrocyte membranes were irradiated at -45 to $-50^{\circ}C$ with an increasing dose of 1.5 MeV electron beam, and after thawing, cytochalasin B binding activities were assayed. Each activity measured was reduced as a simple exponential function of radiation dose. $D_{37}$, dose appeared to be 6.7 mega rads, from which the target size (radiation sensitive mass) of band 4.5 polypeptide was calculated to be 95,500 daltons. This result with other informations available in literature suggests that band 4.5 polypeptide may exist as a dimer in human erythrocytes.

Target Size of $(Na^++K^+)$-ATPase and $Na^+,\;K^+)$Pump of Human Erythrocytes

하종식,Hah, Jong-Sik,Jung, Chan Y. The Korean Physiological Society 1985 대한생리학회지 Vol.19 No.1

$(Na^++K^+)-ATPase$은 ${\alpha}$ 와 ${\beta}$의 두 subunits로 구성되어 있으며, 분자량이 약 300,000 daltons 정도되는 것으로 보아 ${\alpha}_2{\beta}_2$의 형태로 존재할 것으로 알려져 왔다 한편, 사람 적혈구막에 있는 $Na^+,\;K^+\;Pump$는 glycolytic enzymes과 complex를 이루고 있으리라는 보고도 있다. 우리는 이 실험에서 in situ상태의 사람 적혈구막$(Na^++K^+)-ATPase$의 분자량을 측정하기 위하여, 소위 말하는 ‘Target theory’를 radiation에 의한 ouabain sensitive한 $\Na^+$이동과, intact한 cells과 ghosts에서의 ATP가수분해능력의 inactivation data에 적용하였다. Intact한 cells은 cryoprotective agent의 존재하에서, ghosts는 직접적으로 액화질소의 용기속에 담고 온도를 $-45^{\circ}C$에서 $-50^{\circ}C$로 유지시키면서 1.5 MeV의 electron beam으로 조사한 후에 Pump의 기능내지 효소의 활성도를 측정하여 radiation에 따르는 inactivation의 정도를 측정하였다. 이득 활성도는 radiation의 양에 따라 simple exponential function으로 inactivation되었으며, 이로부터 radiation sensitive volume(target size)를 계산하였다. Target size는 intact한 cells을 사용하였을 경 우$(Na^++K^+)-ATPase$나 $Na^+,\;K^+\;Pump$ 모두 600,000 daltons으로 계산되었으며, 이 값은 만약 cells을 strophanthidin으로 먼저 처치하고 측정하면 약 325,000 daltons으로 감소하였다. Ghosts를 사용했을 경우에도$(Na^++K^+)-ATPase$의 target size는 역시 약 325,000 daltons이었다. 이상의 결과로 미루어 보아 intact한 cells에서는 $(Na^++K^+)-ATPase/Na^+,\;K^+\;Pump$가 $(\alpha\beta)_2$의 dimer 상태로 존재하거나 혹은 $(\alpha\beta)_2$의 monomer에 glycolytic enzymes과 같은 다른 enzymes이 붙어 functional한 구조를 이루고 있는 것이 아닌가 사료된다. 또한 실헐성적은 이러한 dimeric association 혹은 heterocomplex association은 ghost를 만드는 과정에서나 strophanthidin의 처치로 부서질 수 있음을 암시하고 있다. Previous biochemical studies indicate that $(Na^++K^+)-ATPase$ is composed of two subunits, ${\alpha}$ and ${\beta}$, in a form of ${\alpha}_2{\beta}_2$ with a molecular weight of approximately 300,000 daltons. There is also suggestive evidence that the $Na^+$, $K^+$ pump in human erythrocytes occurs in a complex with some glycolytic enzymes. We assessed here in situ assembly size of the $(Na^++K^+)-ATPase$ of human erythrocytes by applying classical target theory to radiation inactivation data of the ouabain-sensitive sodium flux and ATP hydrolysis of intact cells and ghosts. Cells(in the presence of cryoprotective agent) and ghosts were irradiated at $-45^{\circ}C$ to $-50^{\circ}C$ with an increasing dose of a 1.5 MeV electron beam, and after thawing, the pump and/or enzyme activities were assayed. Each activity measured was decreased as a simple exponential function of radiation dose, from which a radiation sensitive volume (target size) was calculated. When intact cells were used, the target size of both $(Na^++K^+)-ATPase$ and $Na^+$, $K^+$ pump was found to be approximately 600,000 daltons. This target size of the ATPase was reduced to approximately 325,000 daltons if the cells were pretreated with strophanthidin. When ghosts were used, the target size of the ATPase was again approximately 325,000 daltons. Our target size measurement suggests that, in intact cells, the $(Na^++K^+)-ATPase/Na^+,K^+$ pump exists either as a dimer of $(\alpha\beta)_2$ which is a functional unit or as a monomer of $(\alpha\beta)_2$ but in tight complex with other enzyme or enzymes. The results also suggest that this dimeric or heterocomplex association is dissociated during ghost preparation and strophanthidin treatment.

인슐린의 포도당 이동 촉진 기전에 관한 연구 -세포내부 미세구조와 Cytochalasin B 결합단백질의 분포-

하종식,Hah, Jong-Sik 대한생리학회 1990 대한생리학회지 Vol.24 No.2

What makes glucose transport function sensitive to insulin in one cell type such as adipocyte, and insensitive in another such as liver cells is unresolved question at this time. Recently it is known that insulin stimulates glucose transport in adipocytes largely by redistributing transporter from the storage pool that is included in a low density microsomal fraction to plasma membrane. Therefore, insulin sensitivity may depend upon the relative distribution of gluscose transporters between the plasma membrane and in an intracellular storage compartment. In hepatocytes, the subcellular distribution of glucose transporter is less well documented. It is thus possible that the apparent insensitivity of the hepatocyte system could be either due to lack of the constitutively maintained, intracellular storage pool of glucose transporter or lack of insulin-mediated transporter translocation mechanism in this cell. In this study, I examined if any intracellular glucose transporter pool exists in hepatocytes and this pool is affected by insulin. The results obtained summarized as followings: 1) Distribution of subcellular fractions of hepatocyte showed that there are $24.9{\pm}1.3%$ of plasma membrane, $36.9{\pm}1.7%$ of nucleus-mitochondria enriched fraction, $23.5{\pm}1.2%$ of lysosomal fraction, $9.6{\pm}1.0%$ of high density microsomal fraction and $4.9{\pm}0.5%$ of low density microsomal fraction. 2) In adipocyte, there were $29.9{\pm}2.6%$ of plasma membrane, $19.4{\pm}1.9%$ of nucleus-mitochondria enriched fraction, $26.7{\pm}1.8%$ of high density microsomal fraction and $23.9{\pm}2.1%$ of low density microsomal fraction. 3) Surface labelling of sodium borohydride revealed that plasma membrane contaminated to lysosomal fraction by $26.8{\pm}2.8%$, high density microsomal fraction by $8.3{\pm}1.3%$ and low density microsomal fraction by $1.7{\pm}0.4%$ respectively. 4) Cytochalasin B bound to all of subcellular fractions with a Kd of $1.0{\times}10^{-6}M$. 5) Photolabelling of cytochalasin B to subcellular fractions occurred on 45 K dalton protein band, a putative glucose transporter and D-glucose inhibited the photolabelling. 6) Insulin didn't affect on the distribution of subcellular fractions and translocation of intracellular glucose transporters of hepatocytes. 7) HEGT reconstituted into hepatocytes was largely associated with plasma membrane and very little was found in low density microsomal fraction which equals to the native glucose transporter distribution. Insulin didn't affect on the distribution of exogeneous glucose transporter in hepatocytes. From the above results it is concluded that insulin insensitivity of hepatocyte may due to lack of intracellular storage pool of glucose transporter and thus intracellular storage pool of glucose transporter is an essential feature of the insulin action.

미래 건강부담 추정의 영향요인 고찰 -기후변화에 따른 폭염 증가를 중심으로

하종식 한국환경정책평가연구원 2012 한국환경정책평가연구원 기초연구보고서 Vol.2012 No.-

기초지자체의 환경보건계획 수립 지원시스템과 환경보건상태 평가

하종식,김은채 한국환경보건학회 2022 한국환경보건학회 학술대회 자료집 Vol.2022 No.5

환경포럼 : 환경성질병부담(EBoD) 평가의 국내 개발방안

하종식,신용승 한국환경정책평가연구원 2012 환경포럼 Vol.16 No.12

환경성질병부담(EBoD)은 환경성질환을 유발하는 환경위해인자로부터 야기되는 환경성 위해에 대한 질병부담을 의미한다. 이러한 EBoD는 국가 환경보건정책의 수립, 시행, 그리고 평가에 있어 국가의 환경보건 수준을 대표하는 국가지표로서 유용성이 매우 높다. 본 원고는 국가 환경보건정책 효과에 대한 계량적 지표 또는 정책목표 지표로서 EBoD의 정책 활용성을 높이기 위한 불확실성 평가 및 한국형 EBoD 평가체계의 개발단계를 제안한다. 한국형 EBoD 평가를 도입한다면, EBoD의 정책적 활용성을 고려한 다양한 가정과 가용자료에 대한 체계적인 불확실성 평가 및 한국형 EBoD 평가 방법론 개발, 기초자료 DB 구축, 그리고 관련 환경(보건)정책의 시·공간적 인벤토리 구축을 통한 실질적인 활용도구로의 개발이 필요할 것이다.

Applying policy and health effects of air pollution in South Korea: focus on ambient air quality standards

하종식 환경독성보건학회 2014 환경독성보건학회지 Vol.29 No.-

Objectives South Korea’s air quality standards are insufficient in terms of establishing aprocedure for their management. The current system lacks a proper decision-makingprocess and prior evidence is not considered. The purpose of this study is to propose ameasure for establishing atmospheric environmental standards in South Korea that willtake into consideration the health of its residents. Methods In this paper, the National Ambient Air Quality Standards (NAAQS) of the USwas examined in order to suggest ways, which consider health effects, to establish airquality standards in South Korea. Up-to-date research on the health effects of air pollutionwas then reviewed, and tools were proposed to utilize the key results. This wasdone in an effort to ensure the reliability of the standards with regard to public health. Results This study showed that scientific research on the health effects of air pollutionand the methodology used in the research have contributed significantly to establishingair quality standards. However, as the standards are legally binding, the procedureshould take into account the effects on other sectors. Realistically speaking, it is impossibleto establish standards that protect an entire population from air pollution. Instead,it is necessary to find a balance between what should be done and what can be done. Conclusions Therefore, establishing air quality standards should be done as part of anevidence-based policy that identifies the health effects of air pollution and takes intoconsideration political, economic, and social contexts.