• The Korean Journal of Physiology and Pharmacology
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• 제4권6호
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• pp.497-506
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• 2000

In this work, GLUTs phosphorylations by a downstream effector of PI3-kinase, $PKC-{\zeta},$ were studied, and GLUT4 phosphorylation was compared with GLUT2 phosphorylation in relation to the translocation mechanism. Prior to phosphorylation experiment, $PKC-{\zeta}$ kinase activity was determined as $20.76{\pm}4.09$ pmoles Pi/min/25 ng enzymes. GLUT4 was phosphorylated by $PKC-{\zeta}$ and the phosphorylation was increased on the vesicles immunoadsorpted from LDM and on GLUT4 immunoprecipitated from GLUT4- contianing vesicles of adipocytes treated with insulin. However, GLUT2 in hepatocytes was neither phosphorylated by $PKC-{\zeta}$ nor changed in response to insulin treatment. It was confirmed by measuring the subcellular distribution of GLUT2 based on GLUT2 immunoblot density among the four membrane fractions before and after insulin treatment. Total GLUT2 distributions at PM, LYSO, HDM and LDM were $37.7{\pm}12.0%,\;42.4{\pm}12.1%,\;19.2{\pm}5.0%\;and\;0.7{\pm}1.2%$ in the absence of insulin. Total GLUT2 distribution in the presence of insulin was almost same as that in the absence of insulin. Present data with previous findings suggest that GLUT4 translocation may be attributed to GLUT4 phosphorylation by $PKC-{\zeta}$ but GLUT2 does not translocate because GLUT2 is not phosphorylated by the kinase. Therefore, GLUT phosphorylation may be required in GLUT translocation mechanism.

• The Korean Journal of Physiology and Pharmacology
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• 제4권6호
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• pp.487-496
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• 2000

Insulin stimulates glucose transport in muscle and fat cells by promoting the translocation of glucose transporter (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3-kinase) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt and $PKC-{\zeta}$, those are known as the downstream target of PI3-kinase in regulation of GLUT4 translocation, is not known yet. An interesting possibility is that these protein kinases phosphorylate GLUT4 directly in this process. In the present study, $PKB-{\alpha}$ and $PKC-{\zeta}$ were added exogenously to GLUT4-containing vesicles purified from low density microsome (LDM) of the rat adipocytes by immunoadsorption and immunoprecipitation for direct phosphorylation of GLUT4. Interestingly GLUT4 was phosphorylated by $PKC-{\zeta}$ and its phosphorylation was increased in insulin stimulated state but GLUT4 was not phosphorylated by $PKB-{\alpha}.$ However, the GST-fusion proteins, GLUT4 C-terminal cytoplasmic domain (GLUT4C) and the entire major GLUT4 cytoplasmic domain corresponding to N-terminus, central loop and C-terminus in tandem (GLUT4NLC) were phosphorylated by both $PKB-{\alpha}$ and $PKC-{\zeta}.$ The immunoblots of $PKC-{\zeta}$ and $PKB-{\alpha}$ antibodies with GLUT4-containing vesicles preparation showed that $PKC-{\zeta}$ was co-localized with the vesicles but not $PKB-{\alpha}.$ From the above results, it is clear that $PKC-{\zeta}$ interacts with GLUT4-containing vesicles and it phosphorylates GLUT4 protein directly but $PKB-{\alpha}$ does not interact with GLUT4, suggesting that insulin-elicited signals that pass through PI3-kinase subsequently diverge into two independent pathways, an Akt pathway and a $PKC-{\zeta}$ pathway, and that later pathway contributes, at least in part, insulin stimulation of GLUT4 translocation in adipocytes via a direct GLUT4 phosphorylation.

• 생명과학회지
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• 제23권9호
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• pp.1163-1169
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• 2013

인슐린은 근육세포 표면으로 포도당 수송체 4(glucose transporter 4, GLUT4)를 유도하여 혈액 속의 포도당을 세포 내로 유입시키도록 작용한다고 알려져 있다. Fagopyritol은 인슐린과 유사한 작용을 하는 것으로 알려져 있으므로, 본 연구에서는 혈당강하 효과가 있다고 알려진 fagopyritol을 랫드의 근육세포주(L6GLUT4myc 세포)에 처리하여, 아직 명확하게 밝혀지지 않은 fagopyritol의 혈당강하 기전을 규명하고자 수행하였다. Fagopyritol의 혈당강하 기전을 규명하기 위하여 근원세포(myoblast)와 근관세포(myotube)에 fagopyritol을 처리하여 액틴 필라멘트의 구조와 GLUT4에 미치는 영향을 분석하였다. Fagopyritol을 myoblast에 처리하였을 때, GLUT4가 처리군에서 대조군과 비교하여 유의 있게 원형질막 쪽으로 유도되는 것을 확인하였고, 액틴 필라멘트의 구조가 재조정되면서 GLUT4의 이동을 돕는 것으로 생각된다. 또한 fagopyritol이 인슐린과 유사한 작용 경로를 가지는지 확인하기 위하여, 인슐린 작용 경로에서 중요한 역할을 하는 것으로 알려진 phosphatidylinositol 3-kinase (PI3K)의 억제제인 LY294002를 fagopyritol과 함께 처리하였을 때 GLUT4가 원형질막 쪽으로 유도되지 않는 것을 확인하였다. Fagopyritol을 myotube에 처리하였을 때, myoblast에 처리하였을 때와 유사한 결과를 나타내었다. 이러한 결과를 종합하면 fagopyritol이 인슐린과 유사한 작용을 하여 액틴 필라멘트의 구조 변경과 GLUT4의 이동을 촉진시키는 것으로 사료된다.

• 생명과학회지
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• 제24권8호
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• pp.895-902
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• 2014

아디포넥틴은 이미 합성된 GLUT4의 translocation 증가를 통해 포도당의 세포내 유입을 촉진하며 인슐린 민감도를 증가시키는 것으로 알려져 있다. 본 연구에서는 장기간(6주령부터 16, 26, 36, 47, 및 77주령까지)의 고지방식이(HFD)를 섭취한 비만 C57BL/6 생쥐와, 칼로리제한(CR) 또는 thiazolidinedione (TZD) 섭취에 의해 인슐린 민감성이 회복된 생쥐들로부터 지방조직을 적출하여 아디포넥틴과 GLUT4 의 mRNA 발현의 변화를 조사하였으며, 선형회귀분석(linear regression analysis)을 통해 아디포넥틴과 GLUT4 유전자 발현량 사이의 상관관계를 평가하여 아디포넥틴이 GLUT4 유전자 발현의 전사단계에서도 영향을 미치는지의 가능성을 확인하고자 하였다. 지방조직에서의 유전자 발현량은 TaqMan probe를 이용한 real-time PCR로 정량되었다. 실험결과, 지방조직에서의 아디포넥틴 mRNA발현량은 여러 조건의 생쥐 그룹들 사이에 유의한 변화가 나타나지 않았지만, GLUT4의 유전자 발현량은 HFD군에서는 감소하고, CR군(p<0.05)과 TZD군(p=0.007)에서는 유의하게 증가하는 변화가 확인되었다. 또한, 아디포넥틴과 GLUT4 mRNA 발현량 사이에는 유의한 상관관계를 나타내고 있음이 확인되었다. ND군(p<0.0001), HFD군 p<0.0001), 또는 각각의 주령과 식이별 소그룹, 그리고 CR군(p=0.002) 에서도 두 유전자간의 발현량이 유의하게 연관되어 있었다. 그러나 TZD군(p=0.73)의 생쥐에서는 그 연관성이 사라짐을 관찰하였다. 이는 TZD가 아디포넥틴 유전자 발현에는 영향을 미치지 않지만, GLUT4유전자 발현은 촉진하기에 두 유전자 사이에 유의하지 않은 상관관계로 변화되었음을 시사한다. 이들 결과는 아디포넥틴과 GLUT4의 유전자 발현은 강하게 연관되어 있으며, 두 유전자 발현 조절에 대한 공통적인 작용기전의 존재 가능성 또는 아디포넥틴이 GLUT4 translocation뿐만 아니라 GLUT4의 유전자 발현에도 직접적으로 작용하고 있음을 시사한다.

• Asian-Australasian Journal of Animal Sciences
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• 제23권5호
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• pp.640-648
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• 2010

Insulin-responsive glucose transporter 4 (GLUT4) is a member of the glucose transporter family and mainly presents in skeletal muscle and adipose tissue. To clarify the molecular structure of porcine GLUT4, RACE was used to clone its cDNA. Several cDNA clones corresponding to different regions of GLUT4 were obtained by amplifying reverse-transcriptase products of total RNA extracted from Landrace porcine skeletal muscles. Nucleotide sequence analysis of the cDNA clones revealed that porcine GLUT4 cDNA was composed of 2,491 base pairs with a coding region of 509 amino acids. The deduced amino acid sequence was over 90% identical to human, rabbit and cattle GLUT4. The tissue distribution of GLUT4 was also examined by Real-time RT-PCR. The mRNA expression abundance of GLUT4 was heart>liver, skeletal muscle and brain>lung, kidney and intestine. The developmental expression of GLUT4 and insulin receptor (IR) was also examined by Real-time RT-PCR using total RNA extracted from longissimus dorsi (LM), semimembranosus (SM), and semitendinosus (SD) muscle of Landrace at the age of 1, 7, 30, 60 and 90 d. It was shown that there was significant difference in the mRNA expression level of GLUT4 in skeletal muscles of Landrace at different ages (p<0.05). The mRNA expression level of IR also showed significant difference at different ages (p<0.05). The developmental change in the mRNA expression abundance of GLUT4 was similar to that in IR, and both showed a higher level at birth and 30 d than at other ages. However, there was no significant tissue difference in the mRNA expression of GLUT4 or IR (p>0.05). These results showed that the nucleotide sequence of the cDNA clones was highly identical with human, rabbit and cattle GLUT4 and the developmental change of GLUT4 mRNA in skeletal muscles was similar to that of IR, suggesting that porcine GLUT4 might be an insulin-responsive glucose transporter. Moreover, the tissue distribution of GLUT4 mRNA showed that GLUT4 might be an important nutritional transporter in porcine skeletal muscles.

• The Korean Journal of Physiology
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• 제30권2호
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• pp.231-236
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• 1996

In our previous study (Kim et al, 1991), GLUT 4 protein content correlated negatively with plasma glucose levels in skeletal muscles of STZ-induced diabetic rats. Thus, in this study, to confirm whether expression of GLUT 4 correlate negatively with degree of hyperglycemia, we measured levels of GLUT 4 mRNA in red and white gastrocnemius muscles in STZ-induced mild and severe diabetic rats. Rats were randomly assigned to control, mild, and severe diabetic groups, and the diabetes was induced by intraperitoneal administration of STZ. The experiment was carried out 10 days after STZ administration. Gastrocnemius red and white muscles were used fur the measurement of GLUT 4 expression. Plasma glucose levels of mild and severe diabetic rats were increased compared to control rats (control, mild, and severe diabetes; $6.4{\pm}0.32,\;9.4{\pm}0.68,\;and\;22.0{\pm}0.58$ mmol/L, respectively). Plasma insulin levels of mild and severe diabetic rats were decreased compared to control rats (control, mild, and severe diabetes; $198{\pm}37,\;l14{\pm}14,\;and\;90{\pm}15$ pmol/L, respectively). GLUT 4 mRNA levels of gastrocnemius red muscles in mild and severe diabetic rats were decreased compared to control rats ($64{\pm}1.2%\;and\;71{\pm}2.0%$ of control, respectively), but GLUT 4 mRNA levels in gastrocnemius white muscles were unaltered in diabetic rats. In summary, GLUT 4 mRNA levels were decreased in STZ-induced diabetic rats but did not correlated negatively with degree of hyperglycemia, and this result suggest that the regulatory mechanisms of decreased GLUT 4 mRNA levels are hypoinsulinemia and/or other metabolic factor but not hyperglycemia. And regulation of GLUT 4 expression in STZ-induced diabetes between red and white enriched skeletal muscles may be related to a fiber specific gene regulatory mechanism.

• Archives of Pharmacal Research
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• 제22권4호
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• pp.329-334
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• 1999

Insulin stimulates glucose uptake in muscle and adipose cells primarily by recruiting GLUT4 from an intracellular storage pool to the plasma membrane. Dysfunction of this process known as insulin resistance causes hyperglycemia, a hallmark of diabetes and obesity. Thus the understanding of the mechanisms underlying this process at the molecular level may give an insight into the prevention and treatment of these health problems. GLUT4 in rat adipocytes, for example, constantly recycles between the cells surface and an intracellular pool by endocytosis and exocytosis, each of which is regulated by an insulin-sensitive and GLUT4-selective sorting mechanism. Our working hypothesis has been that this sorting mechanism includes a specific interaction of a cytosolic protein with the GLUT4 cytoplasmic domain. Indeed, a synthetic peptide of the C-terminal cytoplasmic domain of GLUT4 induces an insulin-like GLUT4 recruitment when introduced in rat adipocytes. Relevance of these observations to a novel euglycemic drug design is discussed.

• 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.

• 생명과학회지
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• 제14권5호
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• pp.741-746
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• 2004

본 연구는 운동강도 차이에 따른 카페인 구강 투여가 STZ-유발 당뇨 쥐 가자미근에서 GLUT-4와 GRP-78 단백질 발현에 미치는 영향을 규명하기 위하여 F344계 수컷 횐쥐를 무작위 표본추출에 의하여 당뇨유발군(n=6), 당뇨유발-카페인 투여군(n=6), 당뇨유발-카페인투여 저강도운동군(n=6), 당뇨유발-카페인투여 중강도운동군(n=6), 그리고 당뇨유발-카페인투여 고강도 운동군(n=6)으로 분류하였다. 저강도 운동은 트레드밀 경사도 0%에서 8 m/min 속도로, 중강도 운동은 트레드밀 경사도 0%에서 16 m/min 속도로, 고강도운동은 트레드밀 경사도 0%에서 25 m/min속도로 30분간 1회 운동을 실시하였다. GLUT4단백질 발현은 당뇨군에 비해서 당뇨유발군-카페인 투여군과 당뇨유발-카페인투여 저강도 운동군에서 차이가 없었으며, 당뇨유발-카페인투석 중강도 운동군에서는 다소 감소하였으나 당뇨유발-카페인투여 고강도 운동군에서 증가하였다. GRP-78 단백질 발현은 당뇨군에 비해서 당뇨유발-카페인투여 저강도 운동군, 당뇨유발-카페인투여 중강도 운동군, 그리고 당뇨유발-카페인투석 고강도 운동군에서 감소하였으나, 당뇨유발-카페인 투여군에서는 다소 증가한 것으로 나타났다 고강도 일회성 운동이 인슐린 민감도를 개선시켜 인슐린 요구량을 낮추는데 이러한 효과는 내형질세망에서 세포막으로의 GLUT-4 단백질의 전이와 GLUT-4 단백질 양의 증가 때문이다. 운동군에서의 GRP-78 단백질이 감소된 기전은 정확히 밝힐 수는 없지만, 카페인으로 인한 지질 동원이 운동 시 작업근의 세포에 많은 에너지를 공급하여 세포가 받는 스트레스를 완화시켜 주었기 때문이라고 추측된다.

• Journal of Animal Science and Technology
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• 제47권6호
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• pp.1087-1094
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• 2005

세포 생장과 대사에 있어서 glucose의 세포내 도입은 대부분 동물세포에 필수적이며 이와 같은 도입은 glucose transport protein에 의하여 수행된다. glucose transport protein 중에 GLUT4는 사람과 설치류의 지방조직과 골격근에 있어서 인슐린 자극에 의하여 glucose을 세포내로 도입하는 중요한 역할을 수행한다. 본 연구에서는 한우로부터 이와 같은 GLUT4 유전자를 동정하고 그 발현을 조사하였다. 한우 GLUT4 유전자는 1527bp의 open reading frame으로 구성되어 있으며 509개의 아미노산을 암호화하고 있었다. 그리고 한우 GLUT4 아미노산을 홀스타인, 사람, 생쥐와 비교한 결과 매우 높은 상동성을 나타내었다. 한우 각 조직에 있어서 GLUT4 mRNA의 발현을 확인한 결과 심장에서 가장 높은 발현을 나타내었으며 갈비, 등심, 대장에서는 낮은 발현을 보였다. 그리고 피하지방과 소장지방에서 GLUT4의 발현을 확인하기 위하여 RT-PCR을 수행한 결과 지방조직에서도 발현을 확인할 수 있었다. 한우 intramuscular preadipocyte 세포가 지방세포로 분화하는 과정에 있어서 GLUT4의 발현을 RT-PCR로 확인한 결과 분화에 따라 점차 줄어드는 경향을 나타내었다. 이와 같은 결과는 한우 지방조직에서의 GLUT4 기능은 사람과 생쥐에서의 기능과 다르다는 것을 나타낸다.